Epitope regions of a thyrotrophin (tsh) receptor, uses thereof and antibodies thereto

ABSTRACT

The present invention is concerned with epitope regions of a thyrotrophin (TSH) receptor, uses thereof and antibodies thereto.

This application is a Continuation of U.S. application Ser. No.10/488,074, filed Jun. 17, 2004, which is a National Stage Applicationof PCT/GB2002/03831, filed Aug. 21, 2002, which claims benefit of SerialNo. 0215212.2, filed Jul. 1, 2002 in Great Britain, and also claimsbenefit of Serial No. 0120649.9, filed Aug. 23, 2001 in Great Britainand which application(s) are incorporated herein by reference. A claimof priority to all, to the extent appropriate is made.

The present invention is concerned with epitope regions of athyrotrophin (TSH) receptor, uses thereof and antibodies thereto.

Thyrotrophin or thyroid stimulating hormone (TSH) is a pituitary hormonewhich plays a key role in regulating the function of the thyroid. Itsrelease is stimulated by the hormone TRH formed in the hypothalamus andcontrols the formation and release of the important thyroid hormonesthyroxine (T4) and tri-iodothyronine (T3). On the basis of a feedbackmechanism, the thyroid hormone content of the serum controls the releaseof TSH. The formation of T3 and T4 by the thyroid cells is stimulated byTSH by a procedure in which the TSH released by the pituitary binds tothe TSH receptor of the thyroid cell membrane.

In certain pathological conditions, various types of autoantibodiesagainst this TSH receptor can also be formed. Depending on the type ofthese autoantibodies, either inhibition of the formation and release ofT3 and T4 may occur at the TSH receptor owing to the shielding of theTSH molecules, or, on the other hand, these thyroid hormones may bereleased in an uncontrolled manner because the anti-TSH receptorautoantibodies mimic the action of the TSH and stimulate the synthesisand release of thyroid hormones.

Autoimmune thyroid disease (AMD) is the most common autoimmune diseaseaffecting different populations worldwide. A proportion of patients withAITD, principally those with Graves' disease, have autoantibodies to theTSH receptor substantially as hereinbefore described. The autoantibodiesbind to the TSH receptor and usually mimic the actions of TSH,stimulating the thyroid gland to produce high levels of thyroidhormones. These autoantibodies are described as having stimulatingactivity. In some patients, autoantibodies bind to the TSH receptor butdo not stimulate thyroid hormone production and are described as havingblocking activity [J Sanders, Y Oda, S-A Roberts, M Maruyama, JFurmaniak, B Rees Smith “Understanding the thyrotrophin receptorfunction-structure relationship.” Bailliere's Clinical Endocrinology andMetabolism. Ed. T F Davies 1997 11(3): 451-479. Pub. Bailliere Tindall,London].

Measurements of TSH receptor autoantibodies are important in thediagnosis and management of AMTD, particularly Graves' disease.Currently three types of assays are used to measure TSH receptorautoantibodies:

-   -   (a) competitive binding assays which measure the ability of TSH        receptor autoantibodies to inhibit the binding of TSH to        preparations of TSH receptor;    -   (b) bioassays which measure the ability of TSH receptor        autoantibodies to stimulate cells expressing the TSH receptor in        culture; and    -   (c) immunoprecipitation of TSH receptor preparations with TSH        receptor autoantibodies.

Measurement of TSH receptor autoantibodies using such assays aredescribed in references J Sanders, Y Oda, S-A Roberts, M Maruyama, JFurmaniak, B Rees Smith “Understanding the thyrotrophin receptorfunction-structure relationship.” Bailliere's Clinical Endocrinology andMetabolism. Ed. T F Davies 1997 11(3): 451-479. Pub. Bailliere Tindall,London, and J Sanders, Y Oda, S Roberts, A Kiddie, T Richards, J Bolton,V McGrath, S Walters, D Jaskolski, J Furmaniak, B Rees Smith “Theinteraction of TSH receptor autoantibodies with ¹²⁵I-labelled TSHreceptor.” Journal of Clinical Endocrinology and Metabolism 199984(10):3797-3802.

There are, however, a number of limitations associated with the use ofthe above described currently available assays for measuring TSHreceptor autoantibodies. The competitive assays of type (a) which areavailable in different formats are generally sensitive, relatively easyto perform and adaptable for routine use. However, competitiveradioreceptor assays known to date for detecting TSH receptorautoantibodies have fundamental disadvantages of a practical naturewhich can be ascribed to the fact that the binding ability of TSHreceptor preparations generally react very sensitively to changes in thereceptor or in a biomolecule bound by it. The binding of biomoleculeswhich are peptides or protein in nature, for example hormones orautoantibodies, to receptors is as a rule very complicated in nature,and the specific binding between receptor and bio-molecule is very muchmore sensitive to structural alterations, in particular of the receptor,than is the case with a usual antigen/antibody binding pair which is thebasis of most immunoassays in which receptors are involved. Attempts toimmobilise and/or to label the TSH receptor have as a rule led tostructural alterations which have greatly impaired the functionality ofthe receptor.

As far as bioassays of the type mentioned in (b) are concerned, thesetend to be expensive, time-consuming, require highly skilled staff andare essentially unsuitable for routine use.

With respect to the direct immunoprecipitation assays of type (c),currently available such immunoprecipitation assays do not in practicehave the required sensitivity for TSH receptor autoantibody detection.

The present invention alleviates the problems hitherto associated withthe prior art detection of TSH receptor autoantibodies. Moreparticularly, the present invention provides diagnostic methods and kitsfor screening for TSH receptor autoantibodies, with improved sensitivitycompared to prior art diagnostic methods and kits, and which, ifdesired, allow the use of one or more competitive binding partners orcompetitors for a TSH receptor in competitive assays of the typedescribed above. In particular the present invention is concerned withthe use of one or more identified epitope regions of TSH receptor indiagnostic methods and kits for screening for TSH receptor autoantibodies.

There is provided by the present invention, therefore, for use indiagnosis or therapy of autoimmune disease associated with an immunereaction to a TSH receptor, a polypeptide sequence comprising part orall of the primary structural conformation (that is a continuoussequence of amino acid residues) of one or more TSH receptor epitopeswith which autoantibodies and/or lymphocytes produced in response to aTSH receptor interact (suitably under conditions that allow interactionof a TSH receptor with such autoantibodies or lymphocytes), saidpolypeptide sequence comprising, consisting of or consisting essentiallyof the primary structural conformation of one or more of the following,or one or more variants, analogs, derivatives or fragments thereof, orvariants, analogs or derivatives of such fragments:

-   -   amino acid numbers 22 to 91 of a TSH receptor;    -   amino acid numbers 246 to 260 of a TSH receptor;    -   amino acid numbers 260 to 363 of a TSH receptor; and    -   amino acid numbers 380 to 418 of a TSH receptor;        (in particular said polypeptide sequence comprising, consisting        of or consisting essentially of the primary structural        conformation of amino acid numbers 277 to 296 of a TSH receptor,        or one or more variants, analogs, derivatives or fragments of        amino acid numbers 277 to 296 of a TSH receptor, or variants,        analogs or derivatives of such fragments; and/or the primary        structural conformation of amino acid numbers 246 to 260 of a        TSH receptor, or one or more variants, analogs, derivatives or        fragments of amino acid numbers 246 to 260 of a TSH receptor, or        variants, analogs or derivatives of such fragments);        wherein autoantibodies and/or lymphocytes produced in response        to a TSH receptor interact (suitably under conditions that allow        interaction of a TSH receptor with such autoantibodies or        lymphocytes) with said polypeptide sequence, so as to enable        said diagnosis or therapy.

More particularly, there is provided by the present invention for use indiagnosis or therapy of autoimmune disease associated with an immunereaction to a TSH receptor, a polypeptide sequence comprising,consisting of or consisting essentially of part or all of the primarystructural conformation of one or more TSH receptor epitopes with whichautoantibodies produced in response to a TSH receptor interact (suitablyunder conditions that allow interaction of a TSH receptor with suchautoantibodies), said polypeptide sequence comprising, consisting of orconsisting essentially of the primary structural conformation of one ormore of the following, or one or more variants, analogs, derivatives orfragments thereof, or variants, analogs or derivatives of suchfragments:

-   -   amino acid numbers 22 to 91 of a TSH receptor;    -   amino acid numbers 246 to 260 of a TSH receptor;    -   amino acid numbers 260 to 363 of a TSH receptor; and    -   amino acid numbers 380 to 418 of a TSH receptor;        (in particular said polypeptide sequence comprising, consisting        of or consisting essentially of the primary structural        conformation of amino acid numbers 277 to 296 of a TSH receptor,        or one or more variants, analogs, derivatives or fragments of        amino acid numbers 277 to 296 of a TSH receptor, or variants,        analogs or derivatives of such fragments; and/or the primary        structural conformation of amino acid numbers 246 to 260 of a        TSH receptor, or one or more variants, analogs, derivatives or        fragments of amino acid numbers 246 to 260 of a TSH receptor, or        variants, analogs or derivatives of such fragments);        wherein autoantibodies produced in response to a TSH receptor        interact (suitably under conditions that allow interaction of a        TSH receptor with such autoantibodies) with said polypeptide        sequence, so as to enable said diagnosis or therapy.

Alternatively, there is provided by the present invention for use indiagnosis or therapy of autoimmune disease associated with an immunereaction to a TSH receptor, a polypeptide sequence comprising,consisting of or consisting essentially of part or all of the primarystructural conformation of one or more TSH receptor epitopes with whichlymphocytes produced in response to a TSH receptor interact (suitablyunder conditions that allow interaction of a TSH receptor with suchlymphocytes), said polypeptide sequence comprising, consisting of orconsisting essentially of the primary structural conformation of one ormore of the following, or one or more variants, analogs, derivatives orfragments thereof or variants, analogs or derivatives of such fragments:

-   -   amino acid numbers 22 to 91 of a TSH receptor;    -   amino acid numbers 246 to 260 of a TSH receptor;    -   amino acid numbers 260 to 363 of a TSH receptor; and    -   amino acid numbers 380 to 418 of a TSH receptor;        (in-particular said polypeptide sequence comprising, consisting        of or consisting essentially of the primary structural        conformation of amino acid numbers 277 to 296 of a TSH receptor,        or one or more variants, analogs, derivatives or fragments of        amino acid numbers 277 to 296 of a TSH receptor, or variants,        analogs or derivatives of such fragments; and/or the primary        structural conformation of amino acid numbers 246 to 260 of a        TSH receptor, or one or more variants, analogs, derivatives or        fragments of amino acid numbers 246 to 260 of a TSH receptor, or        variants, analogs or derivatives of such fragments);        wherein lymphocytes produced in response to a TSH receptor        interact (suitably under conditions that allow interaction of a        TSH receptor with such lymphocytes) with said polypeptide        sequence, so as to enable said diagnosis or therapy.

The present invention further provides for use in diagnosis or therapyof autoimmune disease associated with an immune reaction to a TSHreceptor, a polypeptide sequence comprising, consisting of or consistingessentially of part or all of the primary structural conformation of oneor more TSH receptor epitopes with which autoantibodies and/orlymphocytes produced in response to a TSH receptor interact (suitablyunder conditions that allow interaction of a TSH receptor with suchautoantibodies or lymphocytes), said polypeptide sequence comprising,consisting of or consisting essentially of the primary structuralconformation of one or more of the following, or one or more variants,analogs, derivatives or fragments thereof, or variants, analogs orderivatives of such fragments:

-   -   amino acid numbers 22 to 91 of a TSH receptor;    -   amino acid numbers 246 to 260 of a TSH receptor;    -   amino acid numbers 260 to 363 of a TSH receptor; and    -   amino acid numbers 380 to 418 of a TSH receptor;        as depicted in any one of the amino acid sequences of any of        FIGS. 1, 3, 5 and 7, (in particular said polypeptide sequence        comprising, consisting of or consisting essentially of the        primary structural conformation of amino acid numbers 277 to 296        of a TSH receptor as depicted in any one of the amino acid        sequences of FIG. 5, or one or more variants, analogs,        derivatives or fragments of amino acid numbers 277 to 296 of a        TSH receptor as depicted in any one of the amino acid sequences        of FIG. 5, or variants, analogs or derivatives of such        fragments; and/or the primary structural conformation of amino        acid numbers 246 to 260 of a TSH receptor as depicted in any one        of the amino acid sequences of FIG. 3, or one or more variants,        analogs, derivatives or fragments of amino acid numbers 246 to        260 of a TSH receptor as depicted in any one of the amino acid        sequences of FIG. 3, or variants, analogs or derivatives of such        fragments);        wherein autoantibodies and/or lymphocytes produced in response        to a TSH receptor interact (suitably under conditions that allow        interaction of a TSH receptor with such autoantibodies or        lymphocytes) with said polypeptide sequence, so as to enable        said diagnosis or therapy.

More particularly, the present invention further provides for use indiagnosis or therapy of autoimmune disease associated with an immunereaction to a TSH receptor, a polypeptide sequence comprising,consisting of or consisting essentially of part or all of the primarystructural conformation of one or more TSH receptor epitopes with whichautoantibodies produced in response to a TSH receptor interact (suitablyunder conditions that allow interaction of a TSH receptor with suchautoantibodies), said polypeptide sequence comprising, consisting of orconsisting essentially of the primary structural conformation of one ormore of the following, or one or more variants, analogs, derivatives orfragments thereof or variants, analogs or derivatives of such fragments:

amino acid numbers 22 to 91 of a TSH receptor;amino acid numbers 246 to 260 of a TSH receptor;amino acid numbers 260 to 363 of a TSH receptor; andamino acid numbers 380 to 418 of a TSH receptor;as depicted in any one of the amino acid sequences of any of FIGS. 1,3,5 and 7, (in particular said polypeptide sequence comprising, consistingof or consisting essentially of the primary structural conformation ofamino acid numbers 277 to 296 of a TSH receptor as depicted in any oneof the amino acid sequences of FIG. 5, or one or more variants, analogs,derivatives or fragments of amino acid numbers 277 to 296 of a TSHreceptor as depicted in any one of the amino acid sequences of FIG. 5,or variants, analogs or derivatives of such fragments; and/or theprimary structural conformation of amino acid numbers 246 to 260 of aTSH receptor as depicted in any one of the amino acid sequences of FIG.3, or one or more variants, analogs, derivatives or fragments of aminoacid numbers 246 to 260 of a TSH receptor as depicted in any one of theamino acid sequences of FIG. 3, or variants, analogs or derivatives ofsuch fragments);wherein autoantibodies produced in response to a TSH receptor interact(suitably under conditions that allow interaction of a TSH receptor withsuch autoantibodies) with said polypeptide sequence, so as to enablesaid diagnosis or therapy.

The present invention further provides for use in diagnosis or therapyof autoimmune disease associated with an immune reaction to a TSHreceptor, a polypeptide sequence comprising, consisting of or consistingessentially of part or all of the primary structural conformation of oneor more TSH receptor epitopes with which lymphocytes produced inresponse to a TSH receptor interact (suitably under conditions thatallow interaction of a TSH receptor with such lymphocytes), saidpolypeptide sequence comprising, consisting of or consisting essentiallyof the primary structural conformation of one or more of the following,or one or more variants, analogs, derivatives or fragments thereof, orvariants, analogs or derivatives of such fragments:

-   -   amino acid numbers 22 to 91 of a TSH receptor;    -   amino acid numbers 246 to 260 of a TSH receptor;    -   amino acid numbers 260 to 363 of a TSH receptor; and    -   amino acid numbers 380 to 418 of a TSH receptor;        as depicted in any one of the amino acid sequences of any of        FIGS. 1,3, 5 and 7, (in particular said polypeptide sequence        comprising, consisting of or consisting essentially of the        primary structural conformation of amino acid numbers 277 to 296        of a TSH receptor as depicted in any one of the amino acid        sequences of FIG. 5, or one or more variants, analogs,        derivatives or fragments of amino acid numbers 277 to 296 of a        TSH receptor as depicted in any one of the amino acid sequences        of FIG. 5, or variants, analogs or derivatives of such        fragments; and/or the primary structural conformation of amino        acid numbers 246 to 260 of a TSH receptor as depicted in any one        of the amino acid sequences of FIG. 3, or one or more variants,        analogs, derivatives or fragments of amino acid numbers 246 to        260 of a TSH receptor as depicted in any one of the amino acid        sequences of FIG. 3, or variants, analogs or derivatives of such        fragments);        wherein lymphocytes produced in response to a TSH receptor        interact (suitably under conditions that allow interaction of a        TSH receptor with such lymphocytes) with said polypeptide        sequence, so as to enable said diagnosis or therapy.

More preferably, it is generally preferred that such diagnostic ortherapeutic use employs a polypeptide sequence or sequences comprising,consisting of or consisting essentially of the primary structuralconformation of one or more of the following, or one or more variants,analogs, derivatives or fragments thereof, or variants, analogs orderivatives of such fragments:

-   -   amino acid numbers 32 to 41 of a TSH receptor;    -   amino acid numbers 36 to 42 of a TSH receptor;    -   amino acid numbers 247 to 260 of a TSH receptor;    -   amino acid numbers 277 to 296 of a TSH receptor; and    -   amino acid numbers 381 to 385 of a TSH receptor;        (in particular said polypeptide sequence comprising, consisting        of or consisting essentially of the primary structural        conformation of amino acid numbers 277 to 296 of a TSH receptor,        or one or more variants, analogs, derivatives or fragments of        amino acid numbers 277 to 296 of a TSH receptor, or variants,        analogs or derivatives of such fragments; and/or the primary        structural conformation of amino acid numbers 247 to 260 of a        TSH receptor, or one or more variants, analogs, derivatives or        fragments of amino acid numbers 247 to 260 of a TSH receptor, or        variants, analogs or derivatives of such fragments).

In particular, it is generally preferred according to the presentinvention that such diagnostic or therapeutic use employs amino acidnumbers 277 to 296 of a TSH receptor, or one or more variants, analogs,derivatives or fragments thereof, or variants, analogs or derivatives ofsuch fragments.

In particular, it is generally preferred according to the presentinvention that such diagnostic or therapeutic use employs amino acidnumbers 246 to 260 of a TSH receptor, or one or more variants, analogs,derivatives or fragments thereof, or variants, analogs or derivatives ofsuch fragments.

In particular, it is generally preferred according to the presentinvention that such diagnostic or therapeutic use employs amino acidnumbers 247 to 260 of a TSH receptor, or one or more variants, analogs,derivatives or fragments thereof, or variants, analogs or derivatives ofsuch fragments.

A particularly preferred such diagnostic or therapeutic use according tothe present invention, comprises for use in diagnosis or therapy ofautoimmune disease associated with an immune reaction to a TSH receptor:

-   -   (i) a polypeptide sequence comprising, consisting of or        consisting essentially of part or all of the primary structural        conformation of one or more TSH receptor epitopes with which        autoantibodies and/or lymphocytes produced in response to a TSH        receptor interact (suitably under conditions that allow        interaction of a TSH receptor with such autoantibodies or        lymphocytes), said polypeptide sequence comprising, consisting        of or consisting essentially of the primary structural        conformation of amino acid numbers 277 to 296 of a TSH receptor,        or one or more variants, analogs, derivatives or fragments of        amino acid numbers 277 to 296 of a TSH receptor, or variants,        analogs or derivatives of such fragments; and    -   (ii) a polypeptide sequence comprising, consisting of or        consisting essentially of part or all of the primary structural        conformation of one or more further TSH receptor epitopes with        which autoantibodies and/or lymphocytes produced in response to        a TSH receptor interact (suitably under conditions that allow        interaction of a TSH receptor with such autoantibodies or        lymphocytes), said polypeptide sequence comprising, consisting        of or consisting essentially of the primary structural        conformation of amino acid numbers 246 to 260 of a TSH receptor,        or one or more variants, analogs, derivatives or fragments of        amino acid numbers 246 to 260 of a TSH receptor, or variants,        analogs or derivatives of such fragments;        wherein autoantibodies and/or lymphocytes produced in response        to a TSH receptor interact (suitably under conditions that allow        interaction of a TSH receptor with such autoantibodies or        lymphocytes) with said polypeptide sequences, so as to enable        said diagnosis or therapy.

More particularly, such diagnostic or therapeutic use may comprise:

-   -   (i) a polypeptide sequence comprising, consisting of or        consisting essentially of part or all of the primary structural        conformation of one or more TSH receptor epitopes with which        autoantibodies produced in response to a TSH receptor interact        (suitably under conditions that allow interaction of a TSH        receptor with such autoantibodies), said polypeptide sequence        comprising, consisting of or consisting essentially of the        primary structural conformation of amino acid numbers 277 to 296        of a TSH receptor, or one or more variants, analogs, derivatives        or fragments of amino acid numbers 277 to 296 of a TSH receptor,        or variants, analogs or derivatives of such fragments; and    -   (ii) a polypeptide sequence comprising, consisting of or        consisting essentially of part or all of the primary structural        conformation of one or more further TSH receptor epitopes with        which autoantibodies produced in response to a TSH receptor        interact (suitably under conditions that allow interaction of a        TSH receptor with such autoantibodies), said polypeptide        sequence comprising, consisting of or consisting essentially of        the primary structural conformation of amino acid numbers 246 to        260 of a TSH receptor, or one or more variants, analogs,        derivatives or fragments of amino acid numbers 246 to 260 of a        TSH receptor, or variants, analogs or derivatives of such        fragments;        wherein autoantibodies produced in response to a TSH receptor        interact (suitably under conditions that allow interaction of a        TSH receptor with such autoantibodies) with said polypeptide        sequences, so as to enable said diagnosis or therapy.

Alternatively, such diagnostic or therapeutic use may comprise:

-   -   (i) a polypeptide sequence comprising, consisting of or        consisting essentially of part or all of the primary structural        conformation of one or more TSH receptor epitopes with which        lymphocytes produced in response to a TSH receptor interact        (suitably under conditions that allow interaction of a TSH        receptor with such lymphocytes), said polypeptide sequence        comprising, consisting of or consisting essentially of the        primary structural conformation of amino acid numbers 277 to 296        of a TSH receptor, or one or more variants, analogs, derivatives        or fragments of amino acid numbers 277 to 296 of a TSH receptor,        or variants, analogs or derivatives of such fragments; and    -   (ii) a polypeptide sequence comprising, consisting of or        consisting essentially of part or all of the primary structural        conformation of one or more further TSH receptor epitopes with        which lymphocytes produced in response to a TSH receptor        interact (suitably under conditions that allow interaction of a        TSH receptor with such lymphocytes), said polypeptide sequence        comprising, consisting of or consisting essentially of the        primary structural conformation of amino acid numbers 246 to 260        of a TSH receptor, or one or more variants, analogs, derivatives        or fragments of amino acid numbers 246 to 260 of a TSH receptor,        or variants, analogs or derivatives of such fragments;        wherein lymphocytes produced in response to a TSH receptor        interact (suitably under conditions that allow interaction of a        TSH receptor with such lymphocytes) with said polypeptide        sequences, so as to enable said diagnosis or therapy.

A particularly preferred diagnostic or therapeutic use according to thepresent invention, comprises for use in diagnosis or therapy ofautoimmune disease associated with an immune reaction to a TSH receptor:

-   -   (i) a polypeptide sequence comprising, consisting of or        consisting essentially of part or all of the primary structural        conformation of one or more TSH receptor epitopes with which        autoantibodies and/or lymphocytes produced in response to a TSH        receptor interact (suitably under conditions that allow        interaction of a TSH receptor with such autoantibodies or        lymphocytes), said polypeptide sequence comprising, consisting        of or consisting essentially of the primary structural        conformation of amino acid numbers 277 to 296 of a TSH receptor        as depicted in any one of the amino acid sequences of FIG. 5, or        one or more variants, analogs, derivatives or fragments of amino        acid numbers 277 to 296 of a TSH receptor as depicted in any one        of the amino acid sequences of FIG. 5, or variants, analogs or        derivatives of such fragments; and    -   (ii) a polypeptide sequence comprising, consisting of or        consisting essentially of part or all of the primary structural        conformation of one or more filter TSH receptor epitopes with        which autoantibodies and/or lymphocytes produced in response to        a TSH receptor interact (suitably under conditions that allow        interaction of a TSH receptor with such autoantibodies or        lymphocytes), said polypeptide sequence comprising, consisting        of or consisting essentially of the primary structural        conformation of amino acid numbers 246 to 260 of a TSH receptor        as depicted in any one of the amino acid sequences of FIG. 3, or        one or more variants, analogs, derivatives or fragments of amino        acid numbers 246 to 260 of a TSH receptor as depicted in any one        of the amino acid sequences of FIG. 3, or variants, analogs or        derivatives of such fragments;        wherein autoantibodies and/or lymphocytes produced in response        to a TSH receptor interact (suitably under conditions that allow        interaction of a TSH receptor with such autoantibodies or        lymphocytes) with said polypeptide sequences, so as to enable        said diagnosis or therapy.

More particularly, such diagnostic or therapeutic use may comprise:

-   -   (i) a polypeptide sequence comprising, consisting of or        consisting essentially of part or all of the primary structural        conformation of one or more TSH receptor epitopes with which        autoantibodies produced in response to a TSH receptor interact        (suitably under conditions that allow interaction of a TSH        receptor with such autoantibodies), said polypeptide sequence        comprising, consisting of or consisting essentially of the        primary structural conformation of amino acid numbers 277 to 296        of a TSH receptor as depicted in any one of the amino acid        sequences of FIG. 5, or one or more variants, analogs,        derivatives or fragments of amino acid numbers 277 to 296 of a        TSH receptor as depicted in any one of the amino acid sequences        of FIG. 5, or variants, analogs or derivatives of such        fragments; and    -   (ii) a polypeptide sequence comprising, consisting of or        consisting essentially of part or all of the primary structural        conformation of one or more flirter TSH receptor epitopes with        which autoantibodies produced in response to a TSH receptor        interact (suitably under conditions that allow interaction of a        TSH receptor with such autoantibodies), said polypeptide        sequence comprising, consisting of or consisting essentially of        the primary structural conformation of amino acid numbers 246 to        260 of a TSH receptor as depicted in any one of the amino acid        sequences of FIG. 3, or one or more variants, analogs,        derivatives or fragments of amino acid numbers 246 to 260 of a        TSH receptor as depicted in any one of the amino acid sequences        of FIG. 3, or variants, analogs or derivatives of such        fragments;        wherein autoantibodies produced in response to a TSH receptor        interact (suitably under conditions that allow interaction of a        TSH receptor with such autoantibodies) with said polypeptide        sequences, so as to enable said diagnosis or therapy.

Alternatively, such diagnostic or therapeutic use may comprise:

-   -   (i) a polypeptide sequence comprising, consisting of or        consisting essentially of part or all of the primary structural        conformation of one or more TSH receptor epitopes with which        lymphocytes produced in response to a TSH receptor interact        (suitably under conditions that allow interaction of a TSH        receptor with such lymphocytes), said polypeptide sequence        comprising, consisting of or consisting essentially of the        primary structural conformation of amino acid numbers 277 to 296        of a TSH receptor as depicted in any one of the amino acid        sequences of FIG. 5, or one or more variants, analogs,        derivatives or fragments of amino acid numbers 277 to 296 of a        TSH receptor as depicted in any one of the amino acid sequences        of FIG. 5, or variants, analogs or derivatives of such        fragments; and    -   (ii) a polypeptide sequence comprising, consisting of or        consisting essentially of part or all of the primary structural        conformation of one or more further TSH receptor epitopes with        which lymphocytes produced in response to a TSH receptor        interact (suitably under conditions that allow interaction of a        TSH receptor with such lymphocytes), said polypeptide sequence        comprising, consisting of or consisting essentially of the        primary structural conformation of amino acid numbers 246 to 260        of a TSH receptor as depicted in any one of the amino acid        sequences of FIG. 3, or one or more variants, analogs,        derivatives or fragments of amino acid numbers 246 to 260 of a        TSH receptor as depicted in any one of the amino acid sequences        of FIG. 3, or variants, analogs or derivatives of such        fragments;        wherein lymphocytes produced in response to a TSH receptor        interact (suitably under conditions that allow interaction of a        TSH receptor with such lymphocytes) with said polypeptide        sequences, so as to enable said diagnosis or therapy.

It may also be further preferred that the above mentioned diagnostic ortherapeutic use employing:

-   -   (i) a polypeptide sequence comprising, consisting of or        consisting essentially of part or all of the primary structural        conformation of one or more TSH receptor epitopes with which        autoantibodies and/or lymphocytes produced in response to a TSH        receptor interact (suitably under conditions that allow        interaction of a TSH receptor with such autoantibodies or        lymphocytes), said polypeptide sequence comprising, consisting        of or consisting essentially of the primary structural        conformation of amino acid numbers 277 to 296 of a TSH receptor,        or one or more variants, analogs, derivatives or fragments of        amino acid numbers 277 to 296 of a TSH receptor, or variants,        analogs or derivatives of such fragments; and    -   (ii) a polypeptide sequence comprising, consisting of or        consisting essentially of part or all of the primary structural        conformation of one or more further TSH receptor epitopes with        which autoantibodies and/or lymphocytes produced in response to        a TSH receptor interact (suitably under conditions that allow        interaction of a TSH receptor with such autoantibodies or        lymphocytes), said polypeptide sequence comprising, consisting        of or consisting essentially of the primary structural        conformation of amino acid numbers 246 to 260 of a TSH receptor,        or one or more variants, analogs, derivatives or fragments of        amino acid numbers 246 to 260 of a TSH receptor, or variants,        analogs or derivatives of such fragments;        further employs:    -   (iii) a polypeptide sequence comprising, consisting of or        consisting essentially of part or all of the primary structural        conformation of one or more further TSH receptor epitopes with        which autoantibodies and/or lymphocytes produced in response to        a TSH receptor interact (suitably under conditions that allow        interaction of a TSH receptor with such autoantibodies or        lymphocytes), said polypeptide sequence comprising, consisting        of or consisting essentially of the primary structural        conformation of amino acid numbers 381 to 385 of a TSH receptor,        or one or more variants, analogs, derivatives or fragments of        amino acid numbers 381 to 385 of a TSH receptor, or variants,        analogs or derivatives of such fragments.

More particularly, such preferred diagnostic or therapeutic use employs:

-   -   (i) a polypeptide sequence comprising, consisting of or        consisting essentially of part or all of the primary structural        conformation of one or more TSH receptor epitopes with which        autoantibodies and/or lymphocytes produced in response to a TSH        receptor interact (suitably under conditions that allow        interaction of a TSH receptor with such autoantibodies or        lymphocytes), said polypeptide sequence comprising, consisting        of or consisting essentially of the primary structural        conformation of amino acid numbers 277 to 296 of a TSH receptor        as depicted in any one of the amino acid sequences of FIG. 5, or        one or more variants, analogs, derivatives or fragments of amino        acid numbers 277 to 296 of a TSH receptor as depicted in any one        of the amino acid sequences of FIG. 5, or variants, analogs or        derivatives of such fragments;    -   (ii) a polypeptide sequence comprising, consisting of or        consisting essentially of part or all of the primary structural        conformation of one or more further TSH receptor epitopes with        which autoantibodies and/or lymphocytes produced in response to        a TSH receptor interact (suitably under conditions that allow        interaction of a TSH receptor with such autoantibodies or        lymphocytes), said polypeptide sequence comprising, consisting        of or consisting essentially of the primary structural        conformation of amino acid numbers 246 to 260 of a TSH receptor        as depicted in any one of the amino acid sequences of FIG. 3, or        one or more variants, analogs, derivatives or fragments of amino        acid numbers 246 to 260 of a TSH receptor as depicted in any one        of the amino acid sequences of FIG. 3, or variants, analogs or        derivatives of such fragments; and    -   (iii) a polypeptide sequence comprising, consisting of or        consisting essentially of part or all of the primary structural        conformation of one or more further TSH receptor epitopes with        which autoantibodies and/or lymphocytes produced in response to        a TSH receptor interact (suitably under conditions that allow        interaction of a TSH receptor with such autoantibodies or        lymphocytes), said polypeptide sequence comprising, consisting        of or consisting essentially of the primary structural        conformation of amino acid numbers 381 to 385 of a TSH receptor        as depicted in any one of the amino acid sequences of FIG. 7, or        one or more variants, analogs, derivatives or fragments of amino        acid numbers 381 to 385 of a TSH receptor as depicted in any one        of the amino acid sequences of FIG. 7, or variants, analogs or        derivatives of such fragments.

As will be appreciated from the accompanying Figures, the abovementioned amino acid sequences can be of human, porcine, bovine, canine,feline, mouse, rat or ovine origin, and the specific amino acidsequences in each of the above mentioned species are hereinafterdescribed in greater detail with reference to FIGS. 1, 3, 5, and 7.

There also provided by the present invention one or more TSH receptorepitopes with which autoantibodies and/or lymphocytes produced inresponse to a TSH receptor interact (suitably under conditions thatallow interaction of a TSH receptor with such autoantibodies orlymphocytes), said one or more TSH receptor epitopes comprising,consisting of or consisting essentially of one or more of the following,or one or more variants, analogs, derivatives or fragments thereof orvariants, analogs or derivatives of such fragments:

-   -   amino acid numbers 22 to 91 of a TSH receptor;    -   amino acid numbers 246 to 260 of a TSH receptor;    -   amino acid numbers 260 to 363 of a TSH receptor; and    -   amino acid numbers 380 to 418 of a TSH receptor;        (in particular amino acid numbers 277 to 296 of a TSH receptor,        or one or more variants, analogs, derivatives or fragments of        amino acid numbers 277 to 296 of a TSH receptor, or variants,        analogs or derivatives of such fragments; or amino acid numbers        246 to 260 of a TSH receptor, or one or more variants, analogs,        derivatives or fragments of amino acid numbers 246 to 260 of a        TSH receptor, or variants, analogs or derivatives of such        fragments).

More particularly, there is provided by the present invention one ormore TSH receptor epitopes with which autoantibodies produced inresponse to a TSH receptor interact (suitably under conditions thatallow interaction of a TSH receptor with such autoantibodies), said oneor more TSH receptor epitopes comprising, consisting of or consistingessentially of one or more of the following, or one or more variants,analogs, derivatives or fragments thereof, or variants, analogs orderivatives of such fragments:

amino acid numbers 22 to 91 of a TSH receptor;

-   -   amino acid numbers 246 to 260 of a TSH receptor;    -   amino acid numbers 260 to 363 of a TSH receptor; and    -   amino acid numbers 380 to 418 of a TSH receptor;        (in particular amino acid numbers 277 to 296 of a TSH receptor,        or one or more variants, analogs, derivatives or fragments of        amino acid numbers 277 to 296 of a TSH receptor, or variants,        analogs or derivatives of such fragments; or amino acid numbers        246 to 260 of a TSH receptor, or one or more variants, analogs,        derivatives or fragments of amino acid numbers 246 to 260 of a        TSH receptor, or variants, analogs or derivatives of such        fragments).

Alternatively, there is provided by the present invention one or moreTSH receptor epitopes with which lymphocytes produced in response to aTSH receptor interact (suitably under conditions that allow interactionof a TSH receptor with such lymphocytes), said one or more TSH receptorepitopes comprising, consisting of or consisting essentially of one ormore of the following, or one or more variants, analogs, derivatives orfragments thereof, or variants, analogs or derivatives of suchfragments:

-   -   amino acid numbers 22 to 91 of a TSH receptor;    -   amino acid numbers 246 to 260 of a TSH receptor;    -   amino acid numbers 260 to 363 of a TSH receptor; and    -   amino acid numbers 380 to 418 of a TSH receptor;        (in particular amino acid numbers 277 to 296 of a TSH receptor,        or one or more variants, analogs, derivatives or fragments of        amino acid numbers 277 to 296 of a TSH receptor, or variants,        analogs or derivatives of such fragments; or amino acid numbers        246 to 260 of a TSH receptor, or one or more variants, analogs,        derivatives or fragments of amino acid numbers 246 to 260 of a        TSH receptor, or variants, analogs or derivatives of such        fragments).

The present invention further provides one or more TSH receptor epitopeswith which autoantibodies and/or lymphocytes produced in response to aTSH receptor interact (suitably under conditions that allow interactionof a TSH receptor with such autoantibodies or lymphocytes), said one ormore TSH receptor epitopes comprising, consisting of or consistingessentially of one or more of the following, or one or more variants,analogs, derivatives or fragments thereof, or variants, analogs orderivatives of such fragments:

-   -   amino acid numbers 22 to 91 of a TSH receptor;    -   amino acid numbers 246 to 260 of a TSH receptor;    -   amino acid numbers 260 to 363 of a TSH receptor; and    -   amino acid numbers 380 to 418 of a TSH receptor;        as depicted in any one of the amino acid sequences of any of        FIGS. 1, 3, 5 and 7, (in particular amino acid numbers 277 to        296 of a TSH receptor as depicted in any one of the amino acid        sequences of FIG. 5, or one or more variants, analogs,        derivatives or fragments of amino acid numbers 277 to 296 of a        TSH receptor as depicted in any one of the amino acid sequences        of FIG. 5, or variants, analogs or derivatives of such        fragments; or amino acid numbers 246 to 260 of a TSH receptor as        depicted in any one of the amino acid sequences of FIG. 3, or        one or more variants, analogs, derivatives or fragments of amino        acid numbers 246 to 260 of a TSH receptor as depicted in any one        of the amino acid sequences of FIG. 3, or variants, analogs or        derivatives of such fragments).

More particularly, the present invention further provides one or moreTSH receptor epitopes with which autoantibodies produced in response toa TSH receptor interact (suitably under conditions that allowinteraction of a TSH receptor with such autoantibodies), said one ormore TSH receptor epitopes comprising, consisting of or consistingessentially of one or more of the following, or one or more variants,analogs, derivatives or fragments thereof or variants, analogs orderivatives of such fragments:

-   -   amino acid numbers 22 to 91 of a TSH receptor;    -   amino acid numbers 246 to 260 of a TSH receptor;    -   amino acid numbers 260 to 363 of a TSH receptor; and    -   amino acid numbers 380 to 418 of a TSH receptor;        as depicted in any one of the amino acid sequences of any of        FIGS. 1, 3, 5 and 7, (in particular amino acid numbers 277 to        296 of a TSH receptor as depicted in any one of the amino acid        sequences of FIG. 5, or one or more variants, analogs,        derivatives or fragments of amino acid numbers 277 to 296 of a        TSH receptor as depicted in any one of the amino acid sequences        of FIG. 5, or variants, analogs or derivatives of such        fragments; or amino acid numbers 246 to 260 of a TSH receptor as        depicted in any one of the amino acid sequences of FIG. 3, or        one or more variants, analogs, derivatives or fragments of amino        acid numbers 246 to 260 of a TSH receptor as depicted in any one        of the amino acid sequences of FIG. 3, or variants, analogs or        derivatives of such fragments).

The present invention further provides one or more TSH receptor epitopeswith which lymphocytes produced in response to a TSH receptor interact(suitably under conditions that allow interaction of a TSH receptor withsuch lymphocytes), said TSH receptor epitopes comprising, consisting ofor consisting essentially of one or more of the following, or one ormore variants, analogs, derivatives or fragments thereof, or variants,analogs or derivatives of such fragments:

-   -   amino acid numbers 22 to 91 of a TSH receptor;    -   amino acid numbers 246 to 260 of a TSH receptor;    -   amino acid numbers 260 to 363 of a TSH receptor; and    -   amino acid numbers 380 to 418 of a TSH receptor;        as depicted in any one of the amino acid sequences of any of        FIGS. 1, 3, 5 and 7, (in particular amino acid numbers 277 to        296 of a TSH receptor as depicted in any one of the amino acid        sequences of FIG. 5, or one or more variants, analogs,        derivatives or fragments of amino acid numbers 277 to 296 of a        TSH receptor as depicted in any one of the amino acid sequences        of FIG. 5, or variants, analogs or derivatives of such        fragments; or amino acid numbers 246 to 260 of a TSH receptor as        depicted in any one of the amino acid sequences of FIG. 3, or        one or more variants, analogs, derivatives or fragments of amino        acid numbers 246 to 260 of a TSH receptor as depicted in any one        of the amino acid sequences of FIG. 3, or variants, analogs or        derivatives of such fragments).

More preferably, it is generally preferred that one or more TSH receptorepitopes comprise one or more of the following, or one or more variants,analogs, derivatives or fragments thereof, or variants, analogs orderivatives of such fragments:

-   -   amino acid numbers 32 to 41 of a TSH receptor;    -   amino acid numbers 36 to 42 of a TSH receptor;    -   amino acid numbers 247 to 260 of a TSH receptor;    -   amino acid numbers 277 to 296 of a TSH receptor; and    -   amino acid numbers 381 to 385 of a TSH receptor;        (in particular amino acid numbers 277 to 296 of a TSH receptor,        or one or more variants, analogs, derivatives or fragments of        amino acid numbers 277 to 296 of a TSH receptor, or variants,        analogs or derivatives of such fragments; or amino acid numbers        247 to 260 of a TSH receptor, or one or more variants, analogs,        derivatives or fragments of amino acid numbers 247 to 260 of a        TSH receptor, or variants, analogs or derivatives of such        fragments).

A particularly preferred TSH receptor epitope according to the presentinvention comprises, consists of or consists essentially of amino acidnumbers 277 to 296 of a TSH receptor, or one or more variants, analogs,derivatives or fragments thereof, or variants, analogs or derivatives ofsuch fragments, with which autoantibodies and/or lymphocytes produced inresponse to a TSH receptor can interact (suitably under conditions thatallow interaction of a TSH receptor with such autoantibodies orlymphocytes).

A particularly preferred TSH receptor epitope according to the presentinvention comprises, consists of or consists essentially of amino acidnumbers 246 to 260 of a TSH receptor, or one or more variants, analogs,derivatives or fragments thereof, or variants, analogs or derivatives ofsuch fragments, with which autoantibodies and/or lymphocytes produced inresponse to a TSH receptor can interact (suitably under conditions thatallow interaction of a TSH receptor with such autoantibodies orlymphocytes).

A particularly preferred TSH receptor epitope according to the presentinvention comprises, consists of or consists essentially of amino acidnumbers 247 to 260 of a TSH receptor, or one or more variants, analogs,derivatives or fragments thereof, or variants, analogs or derivatives ofsuch fragments, with which autoantibodies and/or lymphocytes produced inresponse to a TSH receptor can interact (suitably under conditions thatallow interaction of a TSH receptor with such autoantibodies orlymphocytes).

There is also provided by the present invention a polypeptide with whichautoantibodies and/or lymphocytes produced in response to a TSH receptorcan interact (suitably under conditions that allow interaction of a TSHreceptor with such autoantibodies or lymphocytes) and which comprises,consists of or consists essentially of part or all of the primarystructural conformation of one or more epitopes of a TSH receptor withwhich autoantibodies and/or lymphocytes produced in response to a TSHreceptor can interact (suitably under conditions that allow interactionof a TSH receptor with such autoantibodies or lymphocytes), whichpolypeptide comprises, consists of or consists essentially of theprimary structural conformation of one or more of the following, or oneor more variants, analogs, derivatives or fragments thereof, orvariants, analogs or derivatives of such fragments:

-   -   amino acid numbers 22 to 91 of a TSH receptor;    -   amino acid numbers 246 to 260 of a TSH receptor;    -   amino acid numbers 260 to 363 of a TSH receptor; and    -   amino acid numbers 380 to 418 of a TSH receptor;        (in particular amino acid numbers 277 to 296 of a TSH receptor,        or one or more variants, analogs, derivatives or fragments of        amino acid numbers 277 to 296 of a TSH receptor, or variants,        analogs or derivatives of such fragments; and/or amino acid        numbers 246 to 260 of a TSH receptor, or one or more variants,        analogs, derivatives or fragments of amino acid numbers 246 to        260 of a TSH receptor, or variants, analogs or derivatives of        such fragments), with which autoantibodies and/or lymphocytes        produced in response to a TSH receptor can interact (suitably        under conditions that allow interaction of a TSH receptor with        such autoantibodies or lymphocytes), with the exception of a        full length TSH receptor.

More particularly, there is provided by the present invention apolypeptide with which autoantibodies produced in response to a TSHreceptor can interact (suitably under conditions that allow interactionof a TSH receptor with such autoantibodies) and which comprises,consists of or consists essentially of part or all of the primarystructural conformation (that is a continuous sequence of amino acidresidues) of one or more epitopes of a TSH receptor with whichautoantibodies produced in response to a TSH receptor can interact(suitably under conditions that allow interaction of a TSH receptor withsuch autoantibodies), which polypeptide comprises, consists of orconsists essentially of the primary structural conformation of one ormore of the following, or one or more variants, analogs, derivatives orfragments thereof, or variants, analogs or derivatives of suchfragments:

-   -   amino acid numbers 22 to 91 of a TSH receptor;    -   amino acid numbers 246 to 260 of a TSH receptor;    -   amino acid numbers 260 to 363 of a TSH receptor; and    -   amino acid numbers 380 to 418 of a TSH receptor;        (in particular amino acid numbers 277 to 296 of a TSH receptor,        or one or more variants, analogs, derivatives or fragments of        amino acid numbers 277 to 296 of a TSH receptor, or variants,        analogs or derivatives of such fragments; and/or amino acid        numbers 246 to 260 of a TSH receptor, or one or more variants,        analogs, derivatives or fragments of amino acid numbers 246 to        260 of a TSH receptor, or variants, analogs or derivatives of        such fragments), with which autoantibodies produced in response        to a TSH receptor can interact (suitably under conditions that        allow interaction of a TSH receptor with such autoantibodies),        with the exception of a full length TSH receptor.

Alternatively, there is provided by the present invention a polypeptidewith which lymphocytes produced in response to a TSH receptor caninteract (suitably under conditions that allow interaction of a TSHreceptor with such lymphocytes) and which comprises, consists of orconsists essentially of part or all of the primary structuralconformation (that is a continuous sequence of amino acid residues) ofone or more epitopes of a TSH receptor with which lymphocytes producedin response to a TSH receptor can interact (suitably under conditionsthat allow interaction of a TSH receptor with such lymphocytes), whichpolypeptide comprises, consists of or consists essentially of theprimary structural conformation of one or more of the following, or oneor more variants, analogs, derivatives or fragments thereof, orvariants, analogs or derivatives of such fragments:

-   -   amino acid numbers 22 to 91 of a TSH receptor;    -   amino acid numbers 246 to 260 of a TSH receptor;    -   amino acid numbers 260 to 363 of a TSH receptor; and    -   amino acid numbers 380 to 418 of a TSH receptor;        (in particular amino acid numbers 277 to 296 of a TSH receptor,        or one or more variants, analogs, derivatives or fragments of        amino acid numbers 277 to 296 of a TSH receptor, or variants,        analogs or derivatives of such fragments; and/or amino acid        numbers 246 to 260 of a TSH receptor, or one or more variants,        analogs, derivatives or fragments of amino acid numbers 246 to        260 of a TSH receptor, or variants, analogs or derivatives of        such fragments), with which lymphocytes produced in response to        a TSH receptor can interact (suitably under conditions that        allow interaction of a TSH receptor with such lymphocytes), with        the exception of a full length TSH receptor.

The present invention further provides a polypep tide with whichautoantibodies and/or lymphocytes produced in response to a TSH receptorcan interact (suitably under conditions that allow interaction of a TSHreceptor with such autoantibodies or lymphocytes) and which comprises,consists of or consists essentially of part or all of the primarystructural conformation of one or more epitopes of a TSH receptor withwhich autoantibodies and/or lymphocytes produced in response to a TSHreceptor can interact (suitably under conditions that allow interactionof a TSH receptor with such autoantibodies or lymphocytes), whichpolypeptide comprises, consists of or consists essentially of theprimary structural conformation of one or more of the following, or oneor more variants, analogs, derivatives or fragments thereof or variants,analogs or derivatives of such fragments:

-   -   amino acid numbers 22 to 91 of a TSH receptor;    -   amino acid numbers 246 to 260 of a TSH receptor;    -   amino acid numbers 260 to 363 of a TSH receptor; and    -   amino acid numbers 380 to 418 of a TSH receptor;        as depicted in any one of the amino acid sequences of any of        FIGS. 1, 3, 5 and 7, (in particular amino acid numbers 277 to        296 of a TSH receptor as depicted in any one of the amino acid        sequences of FIG. 5, or one or more variants, analogs,        derivatives or fragments of amino acid numbers 277 to 296 of a        TSH receptor as depicted in any one of the amino acid sequences        of FIG. 5, or variants, analogs or derivatives of such        fragments; and/or amino acid numbers 246 to 260 of a TSH        receptor as depicted in any one of the amino acid sequences of        FIG. 3, or one or more variants, analogs, derivatives or        fragments of amino acid numbers 246 to 260 of a TSH receptor as        depicted in any one of the amino acid sequences of FIG. 3, or        variants, analogs or derivatives of such fragments), with which        autoantibodies and/or lymphocytes produced in response to a TSH        receptor can interact (suitably under conditions that allow        interaction of a TSH receptor with such autoantibodies or        lymphocytes), with the exception of a full length TSH receptor.

More particularly, the present invention further provides a polypeptidewith which autoantibodies produced in response to a TSH receptor caninteract (suitably under conditions that allow interaction of a TSHreceptor with such autoantibodies) and which comprises, consists of orconsists essentially of part or all of the primary structuralconformation of one or more epitopes of a TSH receptor with whichautoantibodies produced in response to a TSH receptor can interact(suitably under conditions that allow interaction of a TSH receptor withsuch autoantibodies), which polypeptide comprises, consists of orconsists essentially of the primary structural conformation of one ormore of the following, or one or more variants, analogs, derivatives orfragments thereof, or variants, analogs or derivatives of suchfragments:

-   -   amino acid numbers 22 to 91 of a TSH receptor;    -   amino acid numbers 246 to 260 of a TSH receptor;    -   amino acid numbers 260 to 363 of a TSH receptor; and    -   amino acid numbers 380 to 418 of a TSH receptor;        as depicted in any one of the amino acid sequences of any of        FIGS. 1, 3, 5 and 7, (in particular amino acid numbers 277 to        296 of a TSH receptor as depicted in any one of the amino acid        sequences of FIG. 5, or one or more variants, analogs,        derivatives or fragments of amino acid numbers 277 to 296 of a        TSH receptor as depicted in any one of the amino acid sequences        of FIG. 5, or variants, analogs or derivatives of such        fragments; and/or amino acid numbers 246 to 260 of a TSH        receptor as depicted in any one of the amino acid sequences of        FIG. 3, or one or more variants, analogs, derivatives or        fragments of amino acid numbers 246 to 260 of a TSH receptor as        depicted in any one of the amino acid sequences of FIG. 3, or        variants, analogs or derivatives of such fragments), with which        autoantibodies produced in response to a TSH receptor can        interact (suitably under conditions that allow interaction of a        TSH receptor with such autoantibodies), with the exception of a        full length TSH receptor.

The present invention further provides a polypeptide with whichlymphocytes produced in response to a TSH receptor can interact(suitably under conditions that allow interaction of a TSH receptor withsuch lymphocytes) and which comprises, consists of or consistsessentially of part or all of the primary structural conformation of oneor more epitopes of a TSH receptor with which lymphocytes produced inresponse to a TSH receptor can interact (suitably under conditions thatallow interaction of a TSH receptor with such lymphocytes), whichpolypeptide comprises, consists of or consists essentially of theprimary structural conformation of one or more of the following, or oneor more variants, analogs, derivatives or fragments thereof or variants,analogs or derivatives of such fragments:

-   -   amino acid numbers 22 to 91 of a TSH receptor,    -   amino acid numbers 246 to 260 of a TSH receptor,    -   amino acid numbers 260 to 363 of a TSH receptor; and    -   amino acid numbers 380 to 418 of a TSH receptor;        as depicted in any one of the amino acid sequences of any of        FIGS. 1, 3, 5 and 7, (in particular amino acid numbers 277 to        296 of a TSH receptor as depicted in any one of the amino acid        sequences of FIG. 5, or one or more variants, analogs,        derivatives or fragments of amino acid numbers 277 to 296 of a        TSH receptor as depicted in any one of the amino acid sequences        of FIG. 5, or variants, analogs or derivatives of such        fragments; and/or amino acid numbers 246 to 260 of a TSH        receptor as depicted in any one of the amino acid sequences of        FIG. 3, or one or more variants, analogs, derivatives or        fragments of amino acid numbers 246 to 260 of a TSH receptor as        depicted in any one of the amino acid sequences of FIG. 3, or        variants, analogs or derivatives of such fragments), with which        lymphocytes produced in response to a TSH receptor can interact        (suitably under conditions that allow interaction of a TSH        receptor with such lymphocytes), with the exception of a full        length TSH receptor.

More preferably, it is generally preferred that a polypeptide accordingto the present invention can comprise part or all of the primarystructural conformation of one or more epitopes of a TSH receptor withwhich autoantibodies and/or lymphocytes produced in response to a TSHreceptor can interact (suitably under conditions that allow interactionof a TSH receptor with such autoantibodies or lymphocytes) and as suchcomprises, consists of or consists essentially of the primary structuralconformation of one or more of the following, or one or more variants,analogs, derivatives or fragments thereof or variants, analogs orderivatives of such fragments, with which autoantibodies and/orlymphocytes produced in response to a TSH receptor can interact(suitably under conditions that allow interaction of a TSH receptor withsuch autoantibodies or lymphocytes):

-   -   amino acid numbers 32 to 41 of a TSH receptor,    -   amino acid numbers 36 to 42 of a TSH receptor,    -   amino acid numbers 247 to 260 of a TSH receptor,    -   amino acid numbers 277 to 296 of a TSH receptor; and    -   amino acid numbers 381 to 385 of a TSH receptor.

Preferably a polypeptide according to the present invention comprises,consists of or consists essentially of, amino acid numbers 277 to 296 ofa TSH receptor, or one or more variants, analogs, derivatives orfragments thereof, or variants, analogs or derivatives of suchfragments.

Preferably a polypeptide according to the present invention comprises,consists of or consists essentially of, amino acid numbers 246 to 260 ofa TSH receptor, or one or more variants, analogs, derivatives orfragments thereof, or variants, analogs or derivatives of suchfragments.

Preferably a polypeptide according to the present invention comprises,consists of or consists essentially of, amino acid numbers 247 to 260 ofa TSH receptor, or one or more variants, analogs, derivatives orfragments thereof, or variants, analogs or derivatives of suchfragments.

It is also preferred according to the present invention that there isprovided a polypeptide with which autoantibodies and/or lymphocytesproduced in response to a TSH receptor can interact (suitably underconditions that allow interaction of a TSH receptor with suchautoantibodies or lymphocytes) and which comprises part or all of theprimary structural conformation of TSH receptor epitopes with whichautoantibodies and/or lymphocytes produced in response to a TSH receptorcan interact (suitably under conditions that allow interaction of a TSHreceptor with such autoantibodies or lymphocytes), which polypeptidecomprises, consists of or consists essentially of:

-   -   (i) the primary structural conformation of amino acid numbers        277 to 296 of a TSH receptor, or one or more variants, analogs,        derivatives or fragments of amino acid numbers 277 to 296 of a        TSH receptor, or variants, analogs or derivatives of such        fragments, with which autoantibodies and/or lymphocytes produced        in response to a TSH receptor can interact (suitably under        conditions that allow interaction of a TSH receptor with such        autoantibodies or lymphocytes); and    -   (ii) the primary structural conformation of amino acid numbers        246 to 260 of a TSH receptor, or one or more variants, analogs,        derivatives or fragments of amino acid numbers 246 to 260 of a        TSH receptor, or variants, analogs or derivatives of such        fragments, with which autoantibodies and/or lymphocytes produced        in response to a TSH receptor can interact (suitably under        conditions that allow interaction of a TSH receptor with such        autoantibodies or lymphocytes);        with the exception of a full length TSH receptor.

More particularly, there is provided by the present invention apolypeptide with which autoantibodies produced in response to a TSHreceptor can interact (suitably under conditions that allow interactionof a TSH receptor with such autoantibodies) and which comprises part orall of the primary structural conformation of TSH receptor epitopes withwhich autoantibodies produced in response to a TSH receptor can interact(suitably under conditions that allow interaction of a TSH receptor withsuch autoantibodies), which polypeptide comprises, consists of orconsists essentially of:

-   -   (i) the primary structural conformation of amino acid numbers        277 to 296 of a TSH receptor, or one or more variants, analogs,        derivatives or fragments of amino acid numbers 277 to 296 of a        TSH receptor, or variants, analogs or derivatives of such        fragments, with which autoantibodies produced in response to a        TSH receptor can interact (suitably under conditions that allow        interaction of a TSH receptor with such autoantibodies); and    -   (ii) the primary structural conformation of amino acid numbers        246 to 260 of a TSH receptor, or one or more variants, analogs,        derivatives or fragments of amino acid numbers 246 to 260 of a        TSH receptor, or variants, analogs or derivatives of such        fragments, with which autoantibodies produced in response to a        TSH receptor can interact (suitably under conditions that allow        interaction of a TSH receptor with such autoantibodies);        with the exception of a full length TSH receptor.

Alternatively, there is provided by the present invention a polypeptidewith which lymphocytes produced in response to a TSH receptor caninteract (suitably under conditions that allow interaction of a TSHreceptor with such lymphocytes) and which comprises part or all of theprimary structural conformation of TSH receptor epitopes with whichlymphocytes produced in response to a TSH receptor can interact(suitably under conditions that allow interaction of a TSH receptor withsuch lymphocytes), which polypeptide comprises, consists of or consistsessentially of:

-   -   (i) the primary structural conformation of amino acid numbers        277 to 296 of a TSH receptor, or one or more variants, analogs,        derivatives or fragments of amino acid numbers 277 to 296 of a        TSH receptor, or variants, analogs or derivatives of such        fragments, with which lymphocytes produced in response to a TSH        receptor can interact (suitably under conditions that allow        interaction of a TSH receptor with such lymphocytes); and    -   (ii) the primary structural conformation of amino acid numbers        246 to 260 of a TSH receptor, or one or more variants, analogs,        derivatives or fragments of amino acid numbers 246 to 260 of a        TSH receptor, or variants, analogs or derivatives of such        fragments, with which lymphocytes produced in response to a TSH        receptor can interact (suitably under conditions that allow        interaction of a TSH receptor with such lymphocytes);        with the exception of a full length TSH receptor.

The present invention further provides a polypeptide with whichautoantibodies and/or lymphocytes produced in response to a TSH receptorcan interact (suitably under conditions that allow interaction of a TSHreceptor with such autoantibodies or lymphocytes) and which comprisespart or all of the primary structural conformation of epitopes of a TSHreceptor with which autoantibodies and/or lymphocytes produced inresponse to a TSH receptor can interact (suitably under conditions thatallow interaction of a TSH receptor with such autoantibodies orlymphocytes), which polypeptide comprises, consists of or consistsessentially of:

-   -   (i) the primary structural conformation of amino acid numbers        277 to 296 of a TSH receptor as depicted in any one of the amino        acid sequences of FIG. 5, or one or more variants, analogs,        derivatives or fragments of amino acid numbers 277 to 296 of a        TSH receptor as depicted in any one of the amino acid sequences        of FIG. 5, or variants, analogs or derivatives of such        fragments, with which autoantibodies and/or lymphocytes produced        in response to a TSH receptor can interact (suitably under        conditions that allow interaction of a TSH receptor with such        autoantibodies or lymphocytes); and    -   (ii) the primary structural conformation of amino acid numbers        246 to 260 of a TSH receptor as depicted in any one of the amino        acid sequences of FIG. 3, or one or more variants, analogs,        derivatives or fragments of amino acid numbers 246 to 260 of a        TSH receptor as depicted in any one of the amino acid sequences        of FIG. 3, or variants, analogs or derivatives of such        fragments, with which autoantibodies and/or lymphocytes produced        in response to a TSH receptor can interact (suitably under        conditions that allow interaction of a TSH receptor with such        autoantibodies or lymphocytes);        with the exception of a fill length TSH receptor.

More particularly, the present invention further provides a polypeptidewith which autoantibodies produced in response to a TSH receptor caninteract (suitably under conditions that allow interaction of a TSHreceptor with such autoantibodies) and which comprises part or all ofthe primary structural conformation of TSH rector epitopes with whichautoantibodies produced in response to a TSH receptor can interact(suitably under conditions that allow interaction of a TSH receptor withsuch autoantibodies), which polypeptide comprises, consists of orconsists essentially of:

-   -   (i) the primary structural conformation of amino acid numbers        277 to 296 of a TSH receptor as depicted in any one of the amino        acid sequences of FIG. 5, or one or more variants, analogs,        derivatives or fragments of amino acid numbers 277 to 296 of a        TSH receptor as depicted in any one of the amino acid sequences        of FIG. 5, or variants, analogs or derivatives of such        fragments, with which autoantibodies produced in response to a        TSH receptor can interact (suitably under conditions that allow        interaction of a TSH receptor with such autoantibodies); and    -   (ii) the primary structural conformation of amino acid numbers        246 to 260 of a TSH receptor as depicted in any one of the amino        acid sequences of FIG. 3, or one or more variants, analogs,        derivatives or fragments of amino acid numbers 246 to 260 of a        TSH receptor as depicted in any one of the amino acid sequences        of FIG. 3, or variants, analogs or derivatives of such        fragments, with which autoantibodies produced in response to a        TSH receptor can interact (suitably under conditions that allow        interaction of a TSH receptor with such auto antibodies);        with the exception of a full length TSH receptor.

The present invention further provides a polypeptide with whichlymphocytes produced in response to a TSH receptor can interact(suitably under conditions that allow interaction of a TSH receptor withsuch lymphocytes) and which comprises part or all of the primarystructural conformation of TSH receptor epitopes with which lymphocytesproduced in response to a TSH receptor can interact (suitably underconditions that allow interaction of a TSH receptor with suchlymphocytes), which polypeptide comprises, consists of or consistsessentially of:

-   -   (i) the primary structural conformation of amino acid numbers        277 to 296 of a TSH receptor as depicted in any one of the amino        acid sequences of FIG. 5, or one or more variants, analogs,        derivatives or fragments of amino acid numbers 277 to 296 of a        TSH receptor as depicted in any one of the amino acid sequences        of FIG. 5, or variants, analogs or derivatives of such        fragments, with which lymphocytes produced in response to a TSH        receptor can interact (suitably under conditions that allow        interaction of a TSH receptor with such lymphocytes); and    -   (ii) the primary structural conformation of amino acid numbers        246 to 260 of a TSH receptor as depicted in any one of the amino        acid sequences of FIG. 3, or one or more variants, analogs,        derivatives or fragments of amino acid numbers 246 to 260 of a        TSH receptor as depicted in any one of the amino acid sequences        of FIG. 3, or variants, analogs or derivatives of such        fragments, with which lymphocytes produced in response to a TSH        receptor can interact (suitably under conditions that allow        interaction of a TSH receptor with such lymphocytes);        with the exception of a full length TSH receptor.

It is also preferred according to the present invention that there isprovided a polypeptide with which autoant bodies and/or lymphocytesproduced in response to a TSH receptor can interact (suitably underconditions that allow interaction of a TSH receptor with suchautoantibodies or lymphocytes) and which comprises part or all of theprimary structural conformation of TSH receptor epitopes with whichautoantibodies and/or lymphocytes produced in response to a TSH receptorcan interact (suitably under conditions that allow interaction of a TSHreceptor with such autoantibodies or lymphocytes), which polypeptidecomprises, consists of or consists essentially of:

-   -   (i) the primary structural conformation of amino acid numbers        277 to 296 of a TSH receptor, or one or more variants, analogs,        derivatives or fragments of amino acid numbers 277 to 296 of a        TSH receptor, or variants, analogs or derivatives of such        fragments, with which autoantibodies and/or lymphocytes produced        in response to a TSH receptor can react (suitably under        conditions that allow interaction of a TSH receptor with such        autoantibodies or lymphocytes);    -   (ii) the primary structural conformation of amino acid numbers        246 to 260 of a TSH receptor, or one or more variants, analogs,        derivatives or fragments of amino acid numbers 246 to 260 of a        TSH receptor, or variants, analogs or derivatives of such        fragments, with which autoantibodies and/or lymphocytes produced        in response to a TSH receptor can interact (suitably under        conditions that allow interaction of a TSH receptor with such        autoantibodies or lymphocytes); and    -   (iii) the primary structural conformation of amino acid numbers        381 to 385 of a TSH receptor, or one or more variants, analogs,        derivatives or fragments of amino acid numbers 381 to 385 of a        TSH receptor, or variants, analogs or derivatives of such        fragments, with which autoantibodies and/or lymphocytes produced        in response to a TSH receptor can interact (suitably under        conditions that allow interaction of a TSH receptor with such        autoantibodies or lymphocytes);        with the exception of a full length TSH receptor.

More particularly, the present invention further provides a polypeptidewith which autoantibodies and/or lymphocytes produced in response to aTSH receptor can interact (suitably under conditions that allowinteraction of a TSH receptor with such autoantibodies or lymphocytes)and which comprises part or all of the primary structural conformationof epitopes of a TSH receptor with which autoantibodies and/orlymphocytes produced in response to a TSH receptor can interact(suitably under conditions that allow interaction of a TSH receptor withsuch autoantibodies or lymphocytes), which polypeptide comprises,consists of or consists essentially of:

-   -   (i) the primary structural conformation of amino acid numbers        277 to 296 of a TSH receptor as depicted in any one of the amino        acid sequences of FIG. 5, or one or more variants, analogs,        derivatives or fragments of amino acid numbers 277 to 296 of a        TSH receptor as depicted in any one of the amino acid sequences        of FIG. 5, or variants, analogs or derivatives of such        fragments, with which autoantibodies and/or lymphocytes produced        in response to a TSH receptor can interact (suitably under        conditions that allow interaction of a TSH receptor with such        autoantibodies or lymphocytes);    -   (ii) the primary structural conformation of amino acid numbers        246 to 260 of a TSH receptor as depicted in any one of the amino        acid sequences of FIG. 3, or one or more variants, analogs,        derivatives or fragments of amino acid numbers 246 to 260 of a        TSH receptor as depicted in any one of the amino acid sequences        of FIG. 3, or variants, analogs or derivatives of such        fragments, with which autoantibodies and/or lymphocytes produced        in response to a TSH receptor can interact (suitably under        conditions that allow interaction of a TSH receptor with such        autoantibodies or lymphocytes);    -   (iii) the primary structural conformation of amino acid numbers        381 to 385 of a TSH receptor as depicted in any one of the amino        acid sequences of FIG. 7, or one or more variants, analogs,        derivatives or fragments of amino acid numbers 381 to 385 of a        TSH receptor as depicted in any one of the amino acid sequences        of FIG. 7, or variants, analogs or derivatives of such        fragments, with which autoantibodies and/or lymphocytes produced        in response to a TSH receptor can interact (suitably under        conditions that allow interaction of a TSH receptor with such        autoantibodies or lymphocytes);        with the exception of a full length TSH receptor.

As will be appreciated from the accompanying Figures, such amino acidsequences can be of human, porcine, bovine, canine, feline, mouse, rator ovine origin, and the specific amino acid sequences in each of theabove mentioned species are hereinafter described in greater detail withreference to FIGS. 1, 3, 5, and 7. Suitably, in the case wherepolypeptides according to the second aspect of the present inventioncomprise amino acid sequences corresponding to part or all of theprimary structural conformation of more than one epitope of a TSHreceptor, the respective amino acid sequences corresponding to part orall of the primary structural conformation of respective epitopes may beseparated by linker amino acid sequences so as to preferably provide therespective amino acid sequences in a conformation, arrangement orsequence that resembles or substantially resembles a conformation,arrangement or sequence of amino acids as present in an active site of aTSH receptor, and/or can be effective in providing the above referred torespective amino acid sequences of a TSH receptor in a conformation,arrangement or sequence optimal for interaction with autoantibodiesand/or lymphocytes as described herein.

Preferred polypeptide sequences and polypeptides according to thepresent invention comprise, consist of, or consist essentially of, thespecifically referred to amino acid numbered sequences of a TSH receptoras respectively shown in any of accompanying FIG. 1, 3, 5 or 7. Asindicated above, however, the present invention also covers “variants”,“analogs”, “derivatives” and “fragments” of specific amino acidsequences described herein and the terms “variants”, “analogs”,“derivatives” and “fragments” as used herein when referring topolypeptide sequences and polypeptides according to the presentinvention (such as polypeptides having a primary structural conformationof specified amino acids as described herein with reference to theaccompanying Figures) can be characterised as polypeptide sequences andpolypeptides which retain essentially the same biological function oractivity (in terms of autoantibody and/or lymphocyte interaction asdescribed herein) as polypeptide sequences and polypeptides having aprimary structural conformation of specified amino acids as describedherein with reference to the accompanying Figures. Suitably, variants,analogs, derivatives and fragments, or variants, analogs or derivativesof the fragments as described herein can have a primary structuralconformation of amino acids as seen in the accompanying Figures in whichseveral or a few (such as 5 to 10, 1 to 5 or 1 to 3) amino acid residuesare substituted, deleted or added, in any combination. Especiallypreferred among these are silent substitutions, additions are deletionswhich do not alter or substantially alter the biological activity orfunction of polypeptides according to the present invention asspecifically described above. Conservative substitutions can bepreferred as hereinafter described in greater detail.

More particularly, variants, analogs or derivatives of polypeptideshaving a primary structural conformation of specified amino acids asdescribed herein with reference to the accompanying Figures may be:

-   -   (i) ones in which one or more of the amino acid residues are        substituted with a conserved or non-conserved amino acid residue        (preferably a conserved amino acid residue); or    -   (ii) ones in which one or more of the amino acid resides        includes a substituent group; or    -   (iii) ones which further comprise additional amino acids that        can be effective in providing the above referred to amino acid        numbers of a TSH receptor that are present in a polypeptide of        the present invention in a conformation, arrangement or sequence        that resembles or substantially resembles a conformation,        arrangement or sequence of amino acids as present in an active        site of a TSH receptor, and/or can be effective in providing the        above referred to amino acid numbers of a TSH receptor that are        present in a polypeptide of the present invention in a        conformation, arrangement or sequence optimal for interaction        with autoantibodies and/or lymphocytes as described herein.

Such variants, derivatives and analogs are deemed to be within the scopeof those skilled in the art from the teachings herein.

Typically, variants, analogs or derivatives can be those that vary froma reference (such as polypeptides having a primary structuralconformation of specified amino acids as described herein with referenceto the accompanying Figures) by conservative amino acid substitutions.Such substitutions are those that substitute a given amino acid in apolypeptide by another amino acid of like characteristics. Typicallyseen as conservative substitutions are the replacements, one foranother, among the aliphatic amino acids A, V, L and J; among thehydroxyl residues S and T; among the acidic residues D and E; among theamide residues N and Q; among the basic residues K and R; and among thearomatic residues F and Y.

It may be preferred that variants, analogs or derivatives as provided bythe present invention are ones which further comprise additional aminoacids that can be effective in providing the above referred to aminoacid numbers of a TSH receptor that are present in a polypeptide of thepresent invention in a conformation, arrangement or sequence thatresembles or substantially resembles a conformation, arrangement orsequence of amino acids as present in an active site of a TSH receptor,and/or can be effective in providing the above referred to amino acidnumbers of a TSH receptor that are present in a polypeptide of thepresent invention in a conformation, arrangement or sequence optimal forinteraction with autoantibodies and/or lymphocytes as described herein.

More particularly, the term “fragment” as used herein denotes apolypeptide having an amino acid sequence that entirely is the same aspart but not all of the amino acid sequence of a polypeptide having aprimary structural conformation of specified amino acids—as describedherein with reference to the accompanying Figures, and variants orderivatives thereof and such fragments may be “free standing”, i.e. notpart of or fused to other amino acids or polypeptides, or they may becomprised within a larger polypeptide of which they form a part orregion. As will be appreciated, fragments according to the presentinvention comprise or contain the primary structural conformation ofamino acids present in one or more epitopes of a TSH receptor asdescribed herein so as to be capable of interaction with autoantibodiesand/or lymphocytes as described herein.

Polypeptides of the present invention, therefore, include polypeptideshaving a primary structural conformation of specified amino acids asdescribed herein with reference to the accompanying Figures as well aspolypeptides (namely variants, analogs and derivatives as referred toabove) having at least 70% identity to polypeptides having a primarystructural conformation of specified amino acids as described hereinwith reference to the accompanying Figures, preferably at least 80%identity to the polypeptides having a primary structural conformation ofspecified amino acids as described herein with reference to theaccompanying Figures, and more preferably at least 90% identity topolypeptides having a primary structural conformation of specified aminoacids as described herein with reference to the accompanying Figures andstill more preferably at least 95% identity to polypeptides having aprimary structural conformation of specified amino acids as describedherein with reference to the accompanying Figures and also includesfragments of such polypeptides substantially as referred to above.

A polypeptide according to the present invention is suitably obtainedby, or is obtainable by, expression of a polynucleotide according to thepresent invention substantially as hereinafter described. Alternatively,the polypeptides of the invention can be synthetically produced byconventional peptide synthesisers employing techniques which are wellknown in the art. A polypeptide according to the present invention soobtained can be advantageous in being free from association with othereukaryotic polypeptides or contaminants which might otherwise beassociated therewith in its natural environment.

Polypeptides according to the present invention substantially as hereindescribed can be expressed in various systems generating recombinantproteins. For example, for expression in E coli, cDNA coding for theappropriate polypeptides according to the present invention can becloned into a vector, such as pET22, pMEX8, pGEX2T or pQE81L His or anequivalent. In the case of expression in yeast (for exampleSaccharomyces cerevisiae or Schizosaccharomyces pombe), vectors such aspYES2, pESP2 or pYES2/CT or an equivalent, can be employed. AcMNPV(Bac-N-Blue) vector or an equivalent can be used for expression ininsect cells and pRC/CMV, pcDNA3.1 vectors or an equivalent can be usedfor expression in mammalian cells, such as Chinese Hamster Ovary (CHO)cells. A polypeptide according to the present invention can be expressedas a discrete protein, or as a fusion protein linked to, for example,glutathione S transferase (GST) or poly histidine linker. For a discreteprotein, affinity column chromatography purification using a mousemonoclonal antibody to the relevant part of a polypeptide according tothe present invention coupled to a Sepharose particle can be used. If apolypeptide according to the present invention is fused to GST,glutathione Sepharose chromatography purification can be used to isolatethe fusion protein. Specific proteases can be used to separate GST froma polypeptide according to the present invention and a second round ofglutathione Sepharose chromatography can be used to separate GST from apolypeptide according to the present invention. In the case of peptideslinked to poly histidine linker, the purification can be carried outusing immobilised metal affinity chromatography.

The present invention further provides a process of preparing apolypeptide substantially as hereinbefore described, which processcomprises:

-   -   (i) providing a host cell substantially as hereinbefore        described;    -   (ii) growing the host cell; and    -   (iii) recovering a polypeptide according to the present        invention therefrom.

Recovery of a polypeptide according to the present invention cantypically employ conventional isolation and purification techniques,such as chromatographic separations or immunological separations, knownto one of ordinary skill in the art.

In accordance with a further aspect of the present invention, there isprovided a polynucleotide comprising:

-   -   (i) a nucleotide sequence encoding a polypeptide substantially        as hereinbefore described;    -   (ii) a nucleotide sequence encoding a polypeptide substantially        as hereinbefore described, which polypeptide comprises an amino        acid sequence or sequences of specified amino acid numbers of a        TSH receptor which is or arm defined by reference to any of        FIGS. 1, 3, 5 and 7;    -   (iii) a nucleotide sequence encoding a polypeptide of (ii),        which nucleotide sequence comprises nucleotide bases encoding        the above mentioned specified amino acid numbers of a TSH        receptor which are defined by reference to any of FIGS. 1, 3, 5,        and 7, and which nucleotide bases are defined by reference to        any of FIGS. 2, 4, 6 and 8;    -   (iv) a nucleotide sequence differing from the sequence of (iii)        in codon sequence due to the degeneracy of the genetic code;    -   (v) a nucleotide sequence comprising an allelic variation of the        sequence of (iii);    -   (vi) a nucleotide sequence comprising a fragment of any of the        sequences of (i), (ii), (iii), (iv) or (v); or    -   (vii) a nucleotide sequence which hybridizes under stringent        conditions to any of the sequences of (i), (ii), (iii),        (iv), (v) or (vi).

The nucleotide bases of a polynucleotide according to the presentinvention, encoding the above mentioned epitope regions of a polypeptideaccording to the present invention, can be summarised as follows.

Amino Acid Numbers Nucleotide Numbers 22-91  64-273 32{circumflex over( )}1  94-123 36{circumflex over ( )}2 106-126 246-260 736-780 247-260739-780 260-363  778-1089 277-296 829-888 380-418 1138-1254 381-3851141-1155

Polynucleotides of the present invention may be in the form of DNA,including, for instance, cDNA, synthetic DNA and genomic DNAappropriately obtained by cloning or produced by chemical synthetictechniques or by a combination thereof A preferred embodiment of thepresent invention preferably comprises cDNA or synthetic DNA.

The coding sequence which encodes a polypeptide according to the presentinvention may be identical to the coding sequence of a polynucleotide asreferred to above in (iii) and defined by reference to any of FIGS. 2,4, 6 and 8. It also may be a polynucleotide with a different sequence,which, as a result of the redundancy (degeneracy) of the genetic code,encodes a polypeptide according to the present invention.

The present invention further relates to variants of the herein abovedescribed polynucleotides which encode for polypeptides having a primarystructural conformation of specified amino acids as described hereinwith reference to the accompanying Figures, variants, analogs,derivatives or fragments thereof, or variants, analogs or derivatives ofthe fragments and substantially as hereinbefore described in greaterdetail. A variant of the polynucleotide may be a naturally occurringvariant such as a naturally occurring allelic variant, or it may be avariant that is not known to occur naturally. Such non-naturallyoccurring variants of the polynucleotide may be made by mutagenesistechniques.

Among the variants in this regard are variants that differ from theaforementioned polynucleotides by nucle-otide substitutions, deletionsor additions. The substitutions, deletions or additions may involve oneor more nucleotides. Alterations in the coding regions may produceconservative or non-conservative amino acid substitutions, deletions oradditions, again substantially as hereinbefore described.

Variant polynucleotides according to the present invention are suitablyat least 70% identical over their entire length to a polynucleotideencoding polypeptides having a primary structural conformation ofspecified amino acids as described herein with reference to theaccompanying Figures, and polynucleotides which are complementary to, orhybridise to, such polynucleotides. Alternatively, most highly preferredare polynucleotides that comprise a region that is at least 80%identical over its entire length to a polynucleotide encoding apolypeptides having a primary structural conformation of specified aminoacids as described herein with reference to the accompanying Figures andpolynucleotides which are complementary to, or hybridise to, suchpolynucleotides. In this regard, polynucleotides at least 90% identicalover their entire length to the same are particularly preferred, andamong these particularly preferred polynucleotides, those with at least95% identity are especially preferred. Furthermore, those with at least97% identity are highly preferred among those with at least 95%identity, and among these those with at least 98% identity and at least99% identity are particularly highly preferred, with at least 99%identity being the more preferred.

Substantially as hereinbefore described the present invention furtherrelates to polynucleotides that hybridise to the herein above-describedsequences. In this regard, the present invention especially relates topolynucleotides which hybridise under stringent conditions to the hereinabove-described polynucleotides. As herein used, the term “stringentconditions” means hybridisation will occur only if there is at least 95%and preferably at least 97% complementary identity between thesequences.

The present invention also relates to vectors, which comprise apolynucleotide or polynucleotides of the present invention, host cellswhich are genetically engineered with vectors of the invention and theproduction of polypeptides of the invention by recombinant techniques.

The present invention, therefore, further provides a biologicallyfunctional vector system which carries a polynucleotide substantially ashereinbefore described and which is capable of introducing thepolynucleotide into the genome of a host organism.

Host cells can be genetically engineered to incorporate polynucleotidesand express polypeptides of the present invention and the presentinvention further provides a host cell which is transformed ortransfected with a polynucleotide, or one or more polynucleotides, or avector system, each substantially as herein described. The appropriateDNA sequence may be inserted into the vector by any of a variety ofwell-known and routine techniques.

According to a particularly preferred embodiment of the presentinvention, there is also provided a method of screening forautoantibodies or lymphocytes produced in response to a TSH receptor ina sample of body fluid obtained from a subject (in particular a human)suspected of suffering from, susceptible to, having or recovering fromautoimmune disease associated with an immune reaction to a TSH receptor,said method comprising:

-   -   (a) providing either (i) said sample of body fluid from said        subject or (ii) lymphocytes isolated from said sample;    -   (b) contacting said sample or isolated lymphocytes with a        polypeptide according to the present invention substantially as        hereinbefore described (suitably under conditions that allow        interaction of a TSH receptor with autoantibodies or lymphocytes        produced in response to a TSH receptor) so as to permit said        polypeptide to interact with autoantibodies, or lymphocytes,        produced in response to a TSH receptor, and present in, or        isolated from, said sample; and    -   (c) monitoring the degree, or effect, of interaction of said        polypeptide with either said autoantibodies, or said        lymphocytes, produced in response to a TSH receptor and present        in, or isolated from, said sample, thereby providing an        indication of the presence of said autoantibodies, or said        lymphocytes, in said sample, or isolated from said sample.

Substantially as described above, a method according to the presentinvention is suitable for screening for autoantibodies or lymphocytesproduced in response to a TSH receptor in a sample of body fluidobtained from a subject. A method according to the present inventioncan, however, be particularly adapted for use in screening forautoantibodies produced in response to a TSH receptor in a sample ofbody fluid obtained from a subject substantially as hereinafterdescribed in greater detail.

There is in particular provided by the present invention, therefore, amethod of screening for autoantibodies produced in response to a TSHreceptor in a sample of body fluid obtained from a subject (inparticular a human) suspected of suffering from, susceptible to, havingor recovering from autoimmune disease associated with an immune reactionto a TSH receptor, said method comprising:

-   -   (a) providing said sample of body fluid from said subject;    -   (b) contacting said sample with a polypeptide according to the        present invention substantially as hereinbefore described        (suitably under conditions that allow interaction of a TSH        receptor with autoantibodies produced in response to a TSH        receptor) so as to permit said polypeptide to interact with        autoantibodies produced in response to a TSH receptor and        present in said sample; and    -   (c) monitoring the degree of interaction of said polypeptide        with said autoantibodies produced in response to a TSH receptor        and present in said sample, thereby providing an indication of        the presence of said autoantibodies in said sample.

A method according to the present invention may typically employ acontrol, such as a sample of body fluid from a normal subject, in otherwords a subject known to be without autoimmune disease associated withan immune reaction to a TSH receptor.

A method of screening for autoantibodies to a -TSH receptor according tothe present invention may comprise directly monitoring interaction of(i) autoantibodies to a TSH receptor present in the sample of body fluidfrom the subject and (ii) a polypeptide, as provided by the presentinvention substantially as hereinbefore described, typically byemploying non-competitive sandwich type assay techniques known in theart.

Typically, in a method according to the present invention employingnon-competitive techniques, monitoring of the degree of interaction of(i) autoantibodies to a TSH receptor present in the sample and (ii) apolypeptide according to the present invention substantially ashereinbefore described, can comprise providing labelling means either toa polypeptide according to the present invention substantially ashereinbefore described, or to a binding partner for autoantibodies to aTSH receptor, either of which technique would enable monitoring of theabove described interaction. For example, a method according to thepresent invention may comprise directly or indirectly labelling apolypeptide according to the present invention substantially ashereinbefore described; contacting the thus labelled polypeptide with asample of body fluid being screened for TSH receptor autoantibodies soas to provide a mixture thereof; and adding to the mixture a bindingpartner for autoantibodies to a TSH receptor (such as an anti-IgGreagent) present in the sample of body fluid, so as to causeprecipitation of any complexes of labelled polypeptide and TSH receptorautoantibodies present in the mixture. Alternatively, it may bepreferred that a method according to the present invention furthercomprises adding a labelled binding partner for TSH receptorautoantibodies (such as a labelled anti-IgG reagent, for example proteinA or anti-human IgG, or labelled full length TSH receptor or an epitopethereof) to a mixture obtained by contacting (i) a polypeptide accordingto the present invention substantially as hereinbefore describedimmobilised to a support and (ii) a sample of body fluid being screenedfor autoantibodies to a TSH receptor.

It may alternatively be preferred that a method of screening forautoantibodies to a TSH receptor in the sample of body fluid accordingto the present invention, utilises the principles employed in knowncompetitive assays. For example, a method according to the presentinvention may employ at least one competitor capable of competing withautoantibodies to a TSH receptor in the interaction thereof with apolypeptide according to the present invention substantially ashereinbefore described.

Typically, a competitor as employed in a competitive assay methodaccording to the present invention may comprise one or more antibodies,which may be natural or partly or wholly synthetically produced. Acompetitor as employed in the present invention may alternativelycomprise any other protein (for example TSH) having a binding domain orregion which is capable of competing with autoantibodies to a TSHreceptor in the interaction thereof with a polypeptide according to thepresent invention substantially as hereinbefore described. Preferably,however, a competitor as employed in the present invention comprises amonoclonal, recombinant or polyclonal antibody (especially a monoclonalantibody), capable of competing with TSH receptor autoantibodies in theinteraction thereof with a polypeptide according to the presentinvention substantially as hereinbefore described.

Typically, therefore, a competitive assay method according to thepresent invention may further comprise providing at least onecompetitor, such as a monoclonal or polyclonal antibody, whereby in step(b) of a method as herein described a polypeptide according to thepresent invention substantially as hereinbefore described can interactwith either a competitor, such as a monoclonal or polyclonal antibody,or autoantibodies to a TSH receptor present in said sample.

Typically monitoring in a competitive assay method according to thepresent invention comprises comparing:

-   -   (i) interaction of a polypeptide according to the present        invention substantially as hereinbefore described and one or        more competitors substantially as hereinbefore described        (typically a monoclonal or polyclonal antibody), in the absence        of said sample of body fluid being screened (that is a suspected        disease sample), optionally in the presence of a sample of body        fluid from a normal subject, typically a subject known to be        without autoimmune disease associated with an immune reaction to        a TSH receptor; with    -   (ii) interaction of a polypeptide according to the present        invention substantially as hereinbefore described and one or        more competitors substantially as hereinbefore described        (typically a monoclonal or polyclonal antibody), in the presence        of said sample of body fluid being screened.

Typically, the comparison involves observing a decrease in interactionof a polypeptide according to the present invention substantially ashereinbefore described and the competitor in (ii) compared to (i) so asto provide an indication of the presence of autoantibodies to a TSHreceptor in said sample. Typically, the decrease in interaction can beobserved by directly or indirectly labelling the competitor andmonitoring any change in the interaction of the thus labelled competitorwith a polypeptide according to the present invention substantially ashereinbefore described in the absence and in the presence of a sample ofbody fluid being screened for autoantibodies to a TSH receptor. Suitablya polypeptide according to the present invention substantially ashereinbefore described may be immobilised to facilitate the abovementioned monitoring.

Alternatively, there is also provided by the present invention a methodof screening for autoantibodies to a TSH receptor in a sample of bodyfluid obtained from a subject (in particular a human) suspected ofsuffering from, susceptible to, having or recovering from autoimmunedisease associated with an immune reaction to a TSH receptor, saidmethod comprising:

-   -   (a) providing said sample of body fluid from said subject;    -   (b) contacting said sample with        -   (i) a full length TSH receptor (typically a recombinantly            obtained full length TSH receptor), and        -   (ii) at least one competitor capable of competing with            autoantibodies to a TSH receptor in the interaction thereof            with a polypeptide according to the present invention            substantially as hereinbefore described,            -   (suitably under conditions that allow interaction of a                TSH receptor with autoantibodies to a TSH receptor), so                as to permit said fill length TSH receptor to interact                with either autoantibodies to a TSH receptor present in                said sample, or said competitor; and        -   (c) monitoring the interaction of said full length TSH            receptor with said autoantibodies present in said sample,            thereby providing an indication of the presence of said            autoantibodies to a TSH receptor in said sample.

The full length TSH receptor can typically be of human, porcine, bovine,canine, feline, mouse, rat or ovine origin and more preferably arecombinantly obtained full length TSH receptor. A competitor for use insuch an assay typically comprises a monoclonal or polyclonal antibody(preferably monoclonal) substantially as hereinbefore described.

Suitably a detectable label that can be employed in a method accordingto the present invention can be selected from the group consisting ofenzymic labels, isotopic labels, chemiluminescent labels, fluorescentlabels, dyes and the like.

In the case where an isotopic label (such as ¹²⁵I, ¹⁴C, ³H or ³⁵S) isemployed, monitoring may therefore comprise measuring radioactivitydependent on binding of a polypeptide according to the present inventionsubstantially as hereinbefore described. Radioactivity is generallymeasured using a gamma counter, or liquid scintillation counter.

In the case of a method of screening for lymphocytes according to thepresent invention, it is generally preferred that lymphocytes areinitially isolated from a sample of body fluid from a subject usingtechniques well known to one of ordinary skill in the art, followed bycontact with a polypeptide according to the present invention so as tostimulate the proliferation of the isolated lymphocytes. Monitoring ofthe effect of interaction of a polypeptide according to the presentinvention and such proliferating lymphocytes, typically employs meansknown in the art for monitoring such proliferation of lymphocytes.

According to a further particularly preferred embodiment of the presentinvention, there is provided a kit for screening for autoantibodies orlymphocytes produced in response to a TSH receptor in a sample of bodyfluid obtained from a subject (in particular a human) suspected ofsuffering from, susceptible to, having or recovering from autoimmunedisease associated with an immune reaction to a TSH receptor, said kitcomprising:

-   -   (a) a polypeptide according to the present invention        substantially as hereinbefore described;    -   (b) means for contacting either (i) a sample of body fluid        obtained from said subject, or (ii) lymphocytes isolated from a        sample of body fluid obtained from said subject, with said        polypeptide according to the present invention substantially as        hereinbefore described (suitably under conditions that allow        interaction of a TSH receptor with autoantibodies or lymphocytes        produced in response to a TSH receptor) so as to permit said        polypeptide to interact with autoantibodies, or lymphocytes,        produced in response to a TSH receptor, and present in, or        isolated from, said sample; and    -   (c) means for monitoring the degree, or effect, of interaction        of said polypeptide with either said autoantibodies, or said        lymphocytes, produced in response to a TSH receptor and present        in, or isolated from, said sample, thereby providing an        indication of the presence of said autoantibodies, or        lymphocytes, in said sample or isolated from said sample.

Substantially as described above, a kit according to the presentinvention is suitable for screening for autoantibodies or lymphocytesproduced in response to a TSH receptor in a sample of body fluidobtained from a subject. A kit according to the present invention can,however, be particularly adapted for use in screening for autoantibodiesproduced in response to a TSH receptor in a sample of body fluidobtained from a subject substantially as hereinafter described ingreater detail.

There is in particular provided by the present invention, therefore, akit for screening for autoantibodies produced in response to a TSHreceptor in a sample of body fluid obtained from a subject (inparticular a human) suspected of suffering from, susceptible to, havingor recovering from autoimmune disease associated with an immune reactionto a TSH receptor, said kit comprising:

-   -   (a) a polypeptide according to the present invention        substantially as hereinbefore described;    -   (b) means for contacting a sample of body fluid obtained from        said subject with said polypeptide according to the present        invention substantially as hereinbefore described (suitably        under conditions that allow interaction of a TSH receptor with        autoantibodies produced in response to a TSH receptor) so as to        permit said polypeptide to interact with autoantibodies produced        in response to a TSH receptor and present in said sample; and    -   (c) means for monitoring the degree of interaction of said        polypeptide with said autoantibodies produced in response to a        TSH receptor and present in said sample, thereby providing an        indication of the presence of said autoantibodies in said        sample.

A kit according to the present invention may typically further comprisecontrol means, such as means for providing a sample of body fluid from anormal subject, in other words a subject known to be without autoimmunedisease associated with an immune reaction to a TSH receptor.

A kit for screening for autoantibodies to a TSH receptor according tothe present invention may comprise means for directly monitoringinteraction of (i) autoantibodies to a TSH receptor present in thesample of body fluid from the subject and (ii) a polypeptide, asprovided by the present invention substantially as hereinbeforedescribed, typically comprising non-competitive sandwich type assaymeans known in the art.

Typically, in a kit according to the present invention comprisingnon-competitive assay means, means are provided for monitoring thedegree of interaction of (i) autoantibodies to a TSH receptor present inthe sample and (ii) a polypeptide according to the present inventionsubstantially as hereinbefore described, and can comprise labellingmeans provided either to a polypeptide according to the presentinvention substantially as hereinbefore described, or to a bindingpartner for autoantibodies to a TSH receptor, either of which wouldenable monitoring of the above described interaction. For example, a kitaccording to the present invention may comprise means for directly orindirectly labelling a polypeptide according to the present inventionsubstantially as hereinbefore described; means for contacting the thuslabelled polypeptide with a sample of body fluid being screened for aTSH receptor autoantibodies so as to provide a mixture thereof; abinding partner for autoantibodies to a TSH receptor (such as an anti-Igreagent) present in the sample of body fluid; and means for adding thebinding partner to the mixture so as to cause precipitation of anycomplexes of labelled polypeptide and TSH receptor autoantibodiespresent in the mixture. Alternatively, it may be preferred that a kitaccording to the present invention further comprises a labelled bindingpartner for TSH receptor autoantibodies (such as a labelled anti-IgGreagent, for example protein A or anti-human IgG, or labelled fulllength a TSH receptor or an epitope thereof) and means for adding thelabelled binding partner to a mixture obtained by contacting (i) apolypeptide according to the present invention substantially ashereinbefore described immobilised to a support and (ii) a sample ofbody fluid being screened for autoantibodies to a TSH receptor.

It may alternatively be preferred that a kit for screening forautoantibodies to a TSH receptor in the sample of body fluid accordingto the present invention, comprises known competitive assay means. Forexample, a kit according to the present invention may further compriseat least one competitor capable of competing with autoantibodies to aTSH receptor in the interaction thereof with a polypeptide according tothe present invention substantially as hereinbefore described.

Typically, a competitor as employed in a competitive assay kit accordingto the present invention may comprise one or more antibodies, which maybe natural or partly or wholly synthetically produced. A competitor asemployed in the present invention may alternatively comprise any otherprotein having a binding domain or region which is capable of competingwith autoantibodies to a TSH receptor in the interaction thereof with apolypeptide according to the present invention substantially ashereinbefore described. Preferably, however, a competitor as employed inthe present invention comprises a monoclonal or polyclonal antibody(especially a monoclonal antibody), capable of competing with TSHreceptor autoantibodies in the interaction thereof with a polypeptideaccording to the present invention substantially as hereinbeforedescribed.

Typically, therefore, a competitive assay kit according to the presentinvention may further comprise at least one competitor, such as amonoclonal or polyclonal antibody, whereby a polypeptide according tothe present invention substantially as hereinbefore described caninteract with either a competitor, such as a monoclonal or polyclonalantibody, or autoantibodies to a TSH receptor present in a sample ofbody fluid being screened.

Typically monitoring means in a competitive assay kit according to thepresent invention comprise means for comparing:

-   -   (i) interaction of a polypeptide according to the present        invention substantially as hereinbefore described and one or        more competitors substantially as hereinbefore described        (typically a monoclonal or polyclonal antibody), in the absence        of said sample of body fluid being screened (that is a suspected        disease sample), optionally in the presence of a sample of body        fluid from a normal subject, typically a subject known to be        without autoimmune disease associated with an immune reaction to        a TSH receptor; with    -   (ii) interaction of a polypeptide according to the present        invention substantially as hereinbefore described and one or        more competitors substantially as hereinbefore described        (typically a monoclonal or polyclonal antibody), in the presence        of said sample of body fluid being screened.

Typically, the comparison involves observing a decrease in interactionof a polypeptide according to the present invention substantially ashereinbefore described and the competitor in (ii) compared to (i) so asto provide an indication of the presence of autoantibodies to a TSHreceptor in said sample. Typically, the decrease in interaction can beobserved by directly or indirectly labelling the competitor andmonitoring any change in the interaction of the thus labelled competitorwith a polypeptide according to the present invention substantially ashereinbefore described in the absence and in the presence of a sample ofbody fluid being screened for autoantibodies to a TSH receptor. Suitablya polypeptide according to the present invention substantially ashereinbefore described may be immobilised to facilitate the abovementioned monitoring.

Alternatively, there is also provided by the present invention a kit forscreening for autoantibodies to a TSH receptor in a sample of body fluidobtained from a subject (in particular a human) suspected of sufferingfrom, susceptible to, having or recovering from autoimmune diseaseassociated with an immune reaction to a TSH receptor, said kitcomprising:

-   -   (a) a full length TSH receptor (typically a recombinantly        obtained full length TSH receptor);    -   (b) at least one competitor capable of competing with        autoantibodies to a TSH receptor in the interaction thereof with        a polypeptide according to the present invention substantially        as hereinbefore described,    -   (c) means for contacting said sample of body fluid from said        subject, said fall length TSH receptor and said competitor        (suitably under conditions that allow interaction of a TSH        receptor with autoantibodies to a TSH receptor), so as to permit        said full length TSH receptor to interact with either        autoantibodies to a TSH receptor present in said sample, or said        competitor; and    -   (d) means for monitoring the interaction of said full length TSH        receptor with said autoantibodies present in said sample,        thereby providing an indication of the presence of said        autoantibodies to a TSH receptor in said sample.

The full length TSH receptor can typically be of human, porcine, bovine,canine, feline, mouse, rat or ovine origin and more preferably arecombinantly obtained full length TSH receptor. A competitor for use insuch an assay kit typically comprises a monoclonal or polyclonalantibody (preferably monoclonal) substantially as hereinbeforedescribed.

Suitably a detectable label that can be employed in a kit according tothe present invention can be selected from the group consisting ofenzymic labels, isotopic labels, chemiluminescent labels, fluorescentlabels, dyes and the like.

In the case where an isotopic label (such as ¹²⁵I, ¹⁴C, ³H or ³⁵S) isemployed, monitoring means may therefore comprise means for measuringradioactivity dependent on binding of a polypeptide according to thepresent invention substantially as hereinbefore described. Radioactivityis generally measured using a gamma counter, or liquid scintillationcounter.

In the case of a kit for screening for lymphocytes according to thepresent invention, it is generally preferred that means are provided forinitially isolating lymphocytes from a sample of body fluid from asubject, using techniques well known to one of ordinary skill in theart, and means are also provided for contacting a polypeptide accordingto the present invention with such isolated lymphocytes so as tostimulate proliferation of the latter by the former. Means (again knownto one of ordinary skill in the art) for monitoring the effect ofinteraction of a polypeptide according to the present invention and suchproliferating lymphocytes, are also provided in such a kit according tothe present invention.

It will be appreciated from the foregoing description that the presentinvention provides assay methods and kits for detecting autoantibodies(in particular) or lymphocytes produced in response to a TSH receptor ina sample of body fluid substantially as hereinbefore described. Thedetection of such autoantibodies and/or lymphocytes produced in responseto a TSH receptor in the sample of body fluid (or at least the level ofsuch autoantibodies and/or lymphocytes in the sample) is indicative ofthe presence of autoimmune disease associated with an immune reaction toa TSH receptor in the subject from which the sample was obtained andcan, therefore, enable the diagnosis of the likely onset or presence ofautoimmune disease associated with an immune reaction to a TSH receptor.

There is, therefore, further provided by the present invention a methodof diagnosing the likely onset or presence of autoimmune diseaseassociated with an immune reaction to a TSH receptor in a subject (inparticular a human) suspected of suffering from, susceptible to, havingor recovering from, autoimmune disease associated with an immunereaction to a TSH receptor, the method comprising detectingautoantibodies or lymphocytes produced in response to a TSH receptor ina sample of body fluid from the subject substantially as hereinbeforedescribed, and whereby the detected autoantibodies and/or lymphocytescan provide a diagnosis of the likely onset or presence of autoimmunedisease associated with an immune reaction to a TSH receptor in thesubject.

There is still further provided by the present invention a method ofdelaying or preventing the onset of autoimmune disease associated withan immune reaction to a TSH receptor in an animal subject (in particulara human subject) suspected of suffering from, susceptible to orrecovering from autoimmune disease associated with an immune reaction toa TSH receptor, which method comprises initially detectingautoantibodies or lymphocytes indicative of the onset or presence ofautoimmune disease associated with an immune reaction to a TSH receptorin a sample of body fluid obtained from the subject substantially ashereinbefore described, thereby providing a diagnosis of the likelyonset of autoimmune disease associated with an immune reaction to a TSHreceptor in the subject, and thereafter therapeutically treating thesubject so as to delay the onset and/or prevent autoimmune diseaseassociated with an immune reaction to a TSH receptor.

A polypeptide according to the present invention substantially ashereinbefore described is particularly suitable for use in thetherapeutic treatment of autoimmune disease associated with an immunereaction to a TSH receptor. For example, tolerance can be achieved byadministering a polypeptide according to the present inventionsubstantially as hereinbefore described to a subject (in particular ahuman subject) suspected of suffering from, susceptible to, having orrecovering from autoimmune disease associated with an immune reaction toa TSH receptor.

There is, therefore, further provided by the present invention apharmaceutical composition comprising a polypeptide according to thepresent invention substantially as hereinbefore described, together witha pharmaceutically acceptable carrier, diluent or excipient therefor,wherein the polypeptide can interact with autoantibodies and/orlymphocytes produced in response to a TSH receptor.

The present invention further provides a polypeptide according to thepresent invention substantially as hereinbefore described for use in themanufacture of a medicament for the treatment of Graves' disease.

Compositions or medicaments according to the present invention shouldcontain a therapeutic or prophylactic amount of at least one polypeptideaccording to the present invention in a pharmaceutically-acceptablecarrier. The pharmaceutical carrier can be any compatible, non-toxicsubstance suitable for delivery of the polypeptides to the patient.Sterile water, alcohol, fats, waxes, and inert solids may be used as thecarrier. Pharmaceutically-accept able adjuvants, buffering agents,dispersing agents and the like, may also be incorporated into thepharmaceutical compositions. Such compositions can contain a singlepolypeptide or may contain two or more polypeptides according to thepresent invention.

It may be desirable to couple a polypeptide according to the presentinvention to immunoglobulins, e.g. IgG, or to lymphoid cells from thepatient being treated in order to promote tolerance. Such an approach isdescribed in Bradley-Mullen, Activation of Distinct Subsets of TSuppressor Cells with Type III Pneutnococcal Polysaccharide Coupled toSyngeneic Spleen Cells, in: IMMUNOLOGICAL TOLERANCE TO SELF ANDNON-SELF, Buttisto et al, eds., Annals N.Y. Acad. Sci. Vol. 392, pp156-166, 1982. Alternatively, the polypeptides may be modified tomaintain or enhance binding to the MHC while reducing or eliminatingbinding to the associated T-cell receptor. In this way, the modifiedpolypeptides may compete with natural a TSH receptor to inhibit helperT-cell activation and thus inhibit the immune response. In all cases,care should be taken that administration of the pharmaceuticalcompositions of the present invention ameliorate but do not potentiatethe autoimmune response.

Pharmaceutical compositions according to the present invention areuseful for parenteral administration. Preferably, the compositions willbe administered parenterally, i.e. subcutaneously, intramuscularly, orintravenously. Thus, the invention provides compositions for parenteraladministration to a patient, where the compositions comprise a solutionor dispersion of the polypeptides in an acceptable carrier, as describedabove. The concentration of the polypeptides in the pharmaceuticalcomposition can vary widely, i.e. from less than about 0.1% by weight,usually being at least about 1% by weight to as much as 20% by weight ormore. Typical pharmaceutical compositions for intramuscular injectionwould be made up to contain, for example, 1 ml of sterile buffered waterand 1 to 100/ug of a purified polypeptide of the present invention. Atypical composition for intravenous infusion could be made up to contain100 to 500 ml of sterile Ringer's solution and 100 to 500 mg of apurified polypeptide of the present invention. Actual methods forpreparing parenterally administrable compositions are well known in theart and described in more detail in various sources, including, forexample, Remington's Pharmaceutical Science, 15th Edition, MackPublishing Company, Easton, Pa. (1980).

In addition to using a polypeptide according to the present inventiondirectly in pharmaceutical compositions, it is also possible to use apolypeptide according to the present invention to enhance tolerance to aTSH receptor in a subject suspected of suffering from, susceptible to,having or recovering from autoimmune disease associated with an immunereaction to a TSH receptor, employing the following principles. Moreparticularly, peripheral blood lymphocytes can be collected from thesubject in a conventional manner and stimulated by exposure to apolypeptide according to the present invention, as defined above.Usually, other mitogens and growth enhancers will be present, e.g.,phytohemagglutinin, interleukin 2, and the like. Proliferating T-helpercells may be isolated and cloned, also under the stimulation of apolypeptide according to the present invention. Clones which continue toproliferate may then be used to prepare therapeutic compositions for thesubject. The cloned T-cells may be attenuated, e.g. by exposure toradiation, and administered to the subject in order to induce tolerance.Alternatively, the T-cell receptor or portions thereof may be isolatedby conventional protein purification methods from the cloned T-cells andadministered to the individual. Such immunotherapy methods are describedgenerally in Sinha et al. (1990) Science 248:1380-1388.

In some cases, after a T-helper cell has been cloned as described above,it may be possible to develop therapeutic peptides from the T-cellreceptor, where the peptides would be beneficial for treating a patientpopulation suspected of suffering from, susceptible to, having orrecovering from autoimmune disease associated with an immune reaction toa TSH receptor. In such cases, the T-cell receptor gene may be isolatedand cloned by conventional techniques and peptides based on the receptorproduced by recombinant techniques as described above. Therecombinantly-produced peptides may then be incorporated inpharmaceutical compositions as described above.

There is also provided by the present invention a method of cloninglymphocytes produced in response to a TSH receptor, which methodcomprises:

-   -   providing a source of lymphocytes;    -   contacting the lymphocytes with a polypeptide according to the        present invention substantially as hereinbefore described, so as        to effect proliferation of said lymphocytes; and    -   isolating and cloning the proliferating lymphocytes.

The present invention also provides the use of cloned lymphocytesprepared as above, in the therapeutic treatment of autoimmune diseaseassociated with an immune reaction to a TSH receptor. There is provided,therefore, a pharmaceutical composition comprising cloned lymphocytesprepared as above, together with a pharmaceutically acceptable carrier,diluent or excipient therefor and the use of such cloned lymphocytes inthe manufacture of a medicament for the treatment of autoimmune diseaseassociated with an immune reaction to a TSH receptor, in particularGraves' disease.

There is also provided by the present invention one or more therapeuticagents identified as providing a therapeutic effect by interaction withamino acids comprising part or all of the primary conformation of aminoacids of one or more epitopes of a TSH receptor substantially ashereinbefore described, and the present invention further provides oneor more therapeutic agents for use in therapeutically interacting withamino acids comprising part or all of the primary conformation of aminoacids of one or more epitopes of a TSH receptor substantially ashereinbefore described and as such for use in the therapeutic treatmentof an autoimmune disease associated with an immune reaction to a TSHreceptor.

There is, therefore, still further provided by the present invention amethod of treating autoimmune disease associated with an immune reactionto a TSH receptor in a subject, which method comprises initiallydetecting autoantibodies or lymphocytes produced in response to a TSHreceptor in a sample of body fluid obtained from the subjectsubstantially as hereinbefore described, thereby providing a diagnosisof autoimmune disease in the subject, and administering to the subject atherapeutically effective amount of at least one therapeutic agenteffective in the treatment of such autoimmune disease, such as apolypeptide according to the present invention substantially ashereinbefore described.

The present invention also provides a method of treating autoimmunedisease associated with an immune reaction to a TSH receptor in asubject (in particular a human subject), which method comprisesadministering to the subject a therapeutically effective amount of atherapeutic agent identified as providing a therapeutic effect byinteraction with amino acids comprising part or all of the primaryconformation of amino acids of one or more epitopes of a TSH receptorsubstantially as hereinbefore described

The amount of therapeutic agent administered will depend on the specificautoimmune disease state being treated, possibly the age of the patientand will ultimately be at the discretion of an attendant physician.

There is still further provided by the present invention, incombination, a kit substantially as hereinbefore described, togetherwith a therapeutically effective amount of at least one therapeuticagent effective in the treatment of autoimmune disease associated withan immune reaction to a TSH receptor substantially as hereinbeforedescribed.

Substantially as hereinbefore described, the sample of body fluid beingscreened by the present invention will typically comprise blood samplesor other fluid blood fractions, such as in particular serum samples orplasma samples, but the sample may in principle be another biologicalfluid, such as saliva or urine or solubilised tissue extracts, or may beobtained by needle biopsy.

There is still further provided by the present invention a bindingpartner for a TSH receptor, such as an antibody to a TSH receptor, or afragment of an antibody to a TSH receptor, which binding partner caninteract with one or more epitopes to a TSH receptor substantially ashereinbefore described, in particular amino acid numbers 277 to 296 of aTSH receptor. Suitably, antibodies provided by the present invention canbe monoclonal (preferred), recombinant or polyclonal. Typically anantibody, such as a monoclonal antibody, as provided by the presentinvention is in substantially purified form.

More specifically, a monoclonal antibody as provided by the presentinvention can comprise any of monoclonal antibodies 3C7, 2B4, 8E2, 18C5,4D7, 16E5, 17D2, 3B3 and 14D3 or active fragments thereof, as describedin the Examples and further illustrated by the accompanying Figures.Antibodies such as 2B4, 8E2, 1I8C5, 4D7, 16E5, 17D2, 3B3 and 14D3, oractive fragments thereof, as described in the Examples preferably have ahigh affinity for a TSH receptor, such as at least about 108 molar˜1.There is, therefore, further provided by the present invention amonoclonal antibody having an affinity of at least about 10 molar″1 forone or more epitopes of a TSH receptor and which epitope is provided byany one of the following amino acid sequences of a TSH receptor:

-   -   amino acids 22 to 91 of a TSH receptor; or    -   amino acids 246 to 260 of a TSH receptor;        or more particularly, consists essentially of any one of the        following amino acid sequences of a TSH receptor:    -   amino acids 36 to 42 of a TSH receptor; or    -   amino acids 247 to 260 of a TSH receptor.

There is also provided by the present invention a monoclonal antibodyhaving an affinity of at least about 10⁸ molar⁻¹ for one or moreepitopes of a TSH receptor and which epitope is provided by any one ofthe following amino acid sequences of a TSH receptor:

-   -   amino acids 32 to 41 of a TSH receptor; or    -   amino acids 277 to 296 of a TSH receptor.

According to a particularly preferred embodiment of the presentinvention there is provided a binding partner for a TSH receptor, whichbinding partner is capable of binding to a TSH receptor so as tostimulate the TSH receptor, which binding partner does not comprise TSHor naturally produced autoantibodies to the TSH receptor.

Preferably the binding partner comprises an antibody, in particular amonoclonal or recombinant (preferably monoclonal) antibody, capable ofbinding to a TSH receptor so as to stimulate the TSH receptor. Examplesof monoclonal antibodies disclosed herein which stimulate a TSH receptorin this way include 4D7, 16E5, 17D2 and 14D3.

In a preferred case the present invention provides a binding partner fora TSH receptor, which binding partner is capable of binding to the TSHreceptor so as to stimulate the TSH receptor and which comprises:

an antibody V_(H) domain selected from the group consisting of:

-   -   V_(H) domains as shown in any one of FIG. 10, 14, 18, 22, 42, 46        or 50, a V_(H) domain comprising one or more V_(H) CDRs with an        amino acid sequence corresponding to a V_(H) CDR as shown in        FIG. 10, a V_(H) domain comprising one or more V_(H) CDRs with        an amino acid sequence corresponding to a V_(H) CDR as shown in        FIG. 14, a V_(H) domain comprising one or more V_(H) CDRs with        an amino acid sequence corresponding to a V_(H) CDR as shown in        FIG. 18, a V_(H) domain comprising one or more V_(H) CDRs with        an amino acid sequence corresponding to a V_(H) CDR as shown in        FIG. 22, a V_(H) domain comprising one or more V_(H) CDRs with        an amino acid sequence corresponding to a V_(H) CDR as shown in        FIG. 42, a V_(H) domain comprising one or more V_(H) CDRs with        an amino acid sequence corresponding to a V_(H) CDR as shown in        FIG. 46, and a V_(H) domain comprising one or more V_(H) CDRs        with an amino acid sequence corresponding to a V_(H) CDR as        shown in FIG. 50; and/or        an antibody V_(L) domain selected from the group consisting of:    -   V_(L) domains as shown in any one of FIG. 12, 16, 20, 24, 44, 48        or 52, a V_(L) domain comprising one or more V_(L) CDRs with an        amino acid sequence corresponding to a V_(L) CDR as shown in        FIG. 12, a V_(L) domain comprising one or more V_(L) CDRs with        an amino acid sequence corresponding to a V_(L) CDR as shown in        FIG. 16, a V_(L) domain comprising one or more V_(L) CDRs with        an amino acid sequence corresponding to a V_(L) CDR as shown in        FIG. 20, a V_(L) domain comprising one or more V_(L) CDRs with        an amino acid sequence corresponding to a V_(L) CDR as shown in        FIG. 24, a V_(L) domain comprising one or more V_(L) CDRs with        an amino acid sequence corresponding to a V_(L) CDR as shown in        FIG. 44, a V_(L) domain comprising one or more V_(L) CDRs with        an amino acid sequence corresponding to a V_(L) CDR as shown in        FIG. 48, and a V_(L) domain comprising one or more V_(L) CDRs        with an amino acid sequence corresponding to a V_(L) CDR as        shown in FIG. 52.

It may be preferred according to the present invention that a bindingpartner substantially as hereinbefore described comprises an antibodyV_(H) domain substantially as hereinbefore described paired with anantibody V_(L) domain substantially as hereinbefore described to providean antibody binding site comprising both V_(H) and V_(L) domains for aTSH receptor, although as discussed further an antibody V_(H) domain, oran antibody V_(L) domain, may be independently used to bind a TSHreceptor. It will be appreciated, therefore, that a binding partnersubstantially as hereinbefore described can comprise an antibody V_(H)domain substantially as hereinbefore described in the absence of anantibody V_(L) domain. It will also be appreciated, therefore, that abinding partner substantially as hereinbefore described can comprise anantibody V_(L) domain substantially as hereinbefore described in theabsence of an antibody V_(H) domain Alternatively, a binding partnersubstantially as hereinbefore described can comprise an antibody V_(H)domain paired with an antibody V_(L) domain substantially ashereinbefore described to provide an antibody binding site comprisingboth V_(H) and V_(L) domains for a TSH receptor.

Preferred embodiments according to the present invention can thusinclude a binding partner substantially as hereinbefore describedcomprising an antibody V_(H) domain as shown in FIG. 10 paired with anantibody V_(L) domain as shown in FIG. 12 to provide an antibody bindingsite, comprising both these V_(H) and V_(L) domains for a TSH receptor;or a binding partner substantially as hereinbefore described comprisingan antibody V_(H) domain as shown in FIG. 14 paired with an antibodyV_(L) domain as shown in FIG. 16 to provide an antibody binding site,comprising both these V_(H) and V_(L) domains for a TSH receptor; or abinding partner substantially as hereinbefore described comprising anantibody V_(H) domain as shown in FIG. 18 paired with an antibody V_(L)domain as shown in FIG. 20 to provide an antibody binding sitecomprising both these V_(H) and V_(L) domains for a TSH receptor; or abinding partner substantially as hereinbefore described comprising anantibody V_(H) domain as shown in FIG. 22 paired with an antibody V_(L)domain as shown in FIG. 24 to provide an antibody binding sitecomprising both V_(H) and V_(L) domains for a TSH receptor; or a bindingpartner substantially as hereinbefore described comprising an antibodyV_(H) domain as shown in FIG. 42 paired with an antibody V_(L) domain asshown in FIG. 44 to provide an antibody binding site comprising bothV_(H) and V_(L) domains for a TSH receptor, or a binding partnersubstantially as hereinbefore described comprising an antibody V_(H)domain as shown in FIG. 46 paired with an antibody V_(L) domain as shownin FIG. 48 to provide an antibody binding site comprising both V_(H) andV_(L) domains for a TSH receptor, or a binding partner substantially ashereinbefore described comprising an antibody V_(H) domain as shown inFIG. 50 paired with an antibody V_(L) domain as shown in FIG. 52 toprovide an antibody binding site comprising both V_(H) and V_(L) domainsfor a TSH receptor.

It is further envisaged according to the present invention that V_(H)domains substantially as hereinbefore described may be paired with V_(L)domains other than those specifically described herein. It is alsofurther envisaged according to the present invention that V_(L) domainssubstantially as hereinbefore described may be paired with V_(H) domainsother than those specifically described herein.

According to an alternative embodiment of the present invention there isprovided a binding partner substantially as hereinbefore described for aTSH receptor, which binding partner is capable of binding to the TSHreceptor so as to stimulate the TSH receptor and which can comprise:

an antibody V_(H) domain comprising:

-   -   a V_(H) domain comprising one or more V_(H) CDRs with an amino        acid sequence corresponding to a V_(H) CDR as shown in FIG. 10,        or a V_(H) domain comprising one or more V_(H) CDRs with an        amino acid sequence corresponding to a V_(H) CDR as shown in        FIG. 14, or a V_(H) domain comprising one or more V_(H) CDRs        with an amino acid sequence corresponding to a V_(H) CDR as        shown in FIG. 18, or a V_(H) domain comprising one or more V_(H)        CDRs with an amino acid sequence corresponding to a V_(H) CDR as        shown in FIG. 22, or a V_(H) domain comprising one or more V_(H)        CDRs with an amino acid sequence corresponding to a V_(H) CDR as        shown in FIG. 42, or a V_(H) domain comprising one or more V_(H)        CDRs with an amino acid sequence corresponding to a V_(H) CDR as        shown in FIG. 46, or a V_(H) domain comprising one or more V_(H)        CDRs with an amino acid sequence corresponding to a V_(H) CDR as        shown in FIG. 50; and/or        an antibody V_(L) domain comprising:    -   a V_(L) domain comprising one or more V_(L) CDRS with an amino        acid sequence corresponding to a V_(L) CDR as shown in FIG. 12,        or a V_(L) domain comprising one or more V_(L) CDRs with an        amino acid sequence corresponding to a V_(L) CDR as shown in        FIG. 16, or a V_(L) domain comprising one or more V_(L) CDRs        with an amino acid sequence corresponding to a V_(L) CDR as        shown in FIG. 20, or a V_(L) domain comprising one or more V_(L)        CDRs with an amino acid sequence corresponding to a V_(L) CDR as        shown in FIG. 24, or a V_(L) domain comprising one or more V_(L)        CDRs with an amino acid sequence corresponding to a V_(L) CDR as        shown in FIG. 44, or a V_(L) domain comprising one or more V_(L)        CDRs with an amino acid sequence corresponding to a V_(L) CDR as        shown in FIG. 48, or a V_(L) domain comprising one or more V_(L)        CDRs with an amino acid sequence corresponding to a V_(L) CDR as        shown in FIG. 52.

One or more CDRs as referred to above may be taken from the hereinbeforedescribed V_(H) and V_(L) domains and incorporated into a suitableframework. For example, the amino acid sequence of one or more CDRssubstantially as hereinbefore described may be incorporated intoframework regions of antibodies differing from those specificallydisclosed herein, such antibodies thereby incorporating the one or moreCDRs and being capable of binding to the TSH receptor, preferably tostimulate the TSH receptor substantially as hereinbefore described.Alternatively, a binding partner according to the present invention maycomprise a polypeptide capable of binding to the TSH receptor so as tostimulate the TSH receptor substantially as hereinbefore described andcomprising the primary structural conformation of amino acids asrepresented by one or more CDRs as specifically described herein,optionally together with further amino acids, which further amino acidsmay enhance the binding affinity of one or more CDRs as described hereinfor a TSH receptor or may have substantially no role in affecting thebinding properties of the polypeptide for a TSH receptor.

Preferably a binding partner according to the present invention includesan antibody. The term “antibody” as used herein describes animmunoglobulin whether natural or partly or wholly syntheticallyproduced. The term also covers any polypeptide having a binding domainwhich is, or is substantially homologous to, an antibody binding domain.Examples of antibodies are the immunoglobulin isotypes and theirisotypic subclasses and fragments which comprise an antigen bindingdomain such as Fab, scFv or the like.

In particular, fragments of antibodies specifically as herein describedform an important aspect of the present invention. In this way, where abinding partner according to the present invention comprises an antibodysubstantially as hereinbefore described, the antibody may comprise anyof the following fragments: (i) the Fab fragment consisting of V_(L),V_(H), C_(L) and C_(H)1 domains; (ii) the Fd fragment consisting of theV_(H) and C_(H)1 domains; (iii) the Fv fragment consisting of the V_(L)and V_(H) domains; (iv) the dAb fragment which consists of a V_(H)domain; (v) isolated CDR regions; (vi) F(ab′)2 fragments, a bivalentfragment comprising two linked Fab fragments; and (vii) single chain Fvmolecules (scFv), wherein a V_(H) domain and a V_(L) domain are linkedby a peptide linker which allows the two domains to associate to form anantigen binding site.

Alternatively, in the case where a binding partner according to thepresent invention comprises an antibody, the antibody may comprise awhole antibody, whereby the antibody includes variable and constantregions, which variable and constant regions can be further illustratedfor the antibodies provided by the present invention by reference to anyof FIG. 9 to 24, or 41 to 52.

The present invention, also encompasses variants, analogs andderivatives of the specific binding partners, antibodies, V_(H) domains,V_(L) domains, CDRs and polypeptides disclosed herein, which variants,analogs and derivatives retain the ability to bind to the TSH receptorso as to stimulate the TSH receptor substantially as hereinbeforedescribed. The terms variants, analogs and derivatives are substantiallyherein before further described in greater detail with respect topolypeptides according to the present invention and what is meant bythese terms as hereinbefore described applies also to variants, analogsand derivatives of the specific binding partners according to thepresent invention.

The present invention also provides a further binding partner capable ofbinding to the TSH receptor so as to stimulate the TSH receptorsubstantially as hereinbefore described, and which further bindingpartner can compete for binding to the TSH receptor with any specificbinding partner disclosed herein, which further binding partner does notcomprise TSH or autoantibodies to a TSH receptor. In particular thisfurther binding partner may comprise a further antibody having a bindingsite for an epitope region of a TSH receptor suitably as hereinbeforedescribed, which further antibody is capable of binding to the TSHreceptor so as to stimulate the TSH receptor substantially ashereinbefore described and can compete for binding to the TSH receptorwith any specific binding partner disclosed herein.

There is also provided by the present invention a polynucleotidecomprising:

-   -   (i) a nucleotide sequence as shown in any of FIG. 25 to 40, or        53 to 64; or parts of such sequences as shown in FIG. 26, 28,        30, 32, 34, 36, 38, 40, 54, 56, 58, 60, 62, or 64, encoding an        amino acid sequence of an antibody V_(H) domain, an antibody        V_(L) domain or CDR as shown in any of FIG. 10, 12, 14, 16, 18,        20, 22, 24, 42, 44, 46, 48, 50 or 52;    -   (ii) a nucleotide sequence encoding a binding partner        substantially as hereinbefore described, or encoding an amino        acid sequence of an antibody V_(H) domain, an antibody V_(L)        domain or CDR of a binding partner substantially as hereinbefore        described;    -   (iii) a nucleotide sequence encoding a binding partner having a        primary structural conformation of amino acids as shown in any        of FIG. 9 to 24 or 41 to 52, or encoding an amino acid sequence        of an antibody V_(H) domain, an antibody V_(L) domain or CDR as        shown in any of FIG. 10, 12, 14, 16, 18, 20, 22, 24, 42, 44, 46,        48, 50 or 52;    -   (iv) a nucleotide sequence differing from any sequence of (i) in        codon sequence due to the degeneracy of the genetic code;    -   (v) a nucleotide sequence comprising an allelic variation of any        sequence of (i);    -   (vi) a nucleotide sequence comprising a fragment of any of the        sequences of (i), (ii), (iii), (iv) or (v), and in particular a        nucleotide sequence comprising a fragment of any of the        sequences of (i), (ii), (iii), (iv) or (v) and encoding a Fab        fragment, aFd fragment, a Fv fragment, a dAb fragment, an        isolated CDR region, F(ab′)2 fragments or a scFv fragment, of a        binding partner substantially as hereinbefore described;    -   (vii) a nucleotide sequence differing from the any sequence        of (i) due to mutation, deletion or substitution of a nucleotide        base and encoding a binding partner substantially as        hereinbefore described, or encoding an amino acid sequence of an        antibody V_(H) domain, an antibody V_(L) domain or CDR of a        binding partner substantially as hereinbefore described.

Variant polynucleotides according to the present invention are suitablyat least 70% identical over their entire length to any polynucleotidesequence of (i), most highly preferred are polynucleotides that comprisea region that is at least 80% identical over its entire length to anypolynucleotide sequence of (i), polynucleotides at least 90% identicalover their entire length to any polynucleotide sequence of (i) areparticularly preferred, and among these particularly preferredpolynucleotides, those with at least 95% identity are especiallypreferred. What is meant by variants of specific polynucleotidesequences described herein is hereinbefore described in greater detail.

The present invention further provides a biologically functional vectorsystem which carries a polynucleotide substantially as hereinbeforedescribed and which is capable of introducing the polynucleotide intothe genome of a host organism.

The present invention also relates to host cells which are transformedwith polynucleotides of the invention and the production of bindingpartners of the invention by recombinant techniques. Host cells can begenetically engineered to incorporate polynucleotides and expressbinding partners of the present invention.

A binding partner substantially as hereinbefore described may havediagnostic and therapeutic applications, and may advantageously interactor bind with one or more epitope regions of a TSH receptor substantiallyas hereinbefore described.

Accordingly, a binding partner substantially as hereinbefore describedcan be employed in screening methods for detecting autoantibodiessubstantially as hereinbefore described and also in diagnostic methodssubstantially as hereinbefore described. In this way, binding partnersaccording to the present invention can be employed in place ofcompetitors hitherto described for use in screening methods fordetecting autoantibodies substantially as hereinbefore described andalso in diagnostic methods substantially as hereinbefore described.Similarly, binding partners according to the present invention can beemployed in place of competitors hitherto described for use in kits foruse in detecting autoantibodies substantially as hereinbefore described.

The present invention also provides a method of screening forautoantibodies to a TSH receptor in a sample of body fluid obtained froma subject suspected of suffering from, susceptible to, having orrecovering from autoimmune disease associated with an immune reaction toa TSH receptor, said method comprising:

-   -   (a) providing said sample of body fluid from said subject;    -   (b) contacting said sample with        -   (i) a full length TSH receptor, one or more epitopes thereof            or a polypeptide comprising one or more epitopes of a TSH            receptor, and        -   (ii) one or more binding partners substantially as            hereinbefore described;        -   (suitably under conditions that allow interaction of a TSH            receptor with autoantibodies produced in response to a TSH            receptor) so as to permit said TSH receptor, said one or            more epitopes thereof or said polypeptide, to interact with            either autoantibodies to a TSH receptor present in said            sample, or said one or more binding partners; and    -   (c) monitoring the interaction of said TSH receptor, said one or        more epitopes thereof or said polypeptide, with said        autoantibodies present in said sample, thereby providing an        indication of the presence of said autoantibodies to a TSH        receptor in said sample.

Preferably, a method according to the present invention as referred toabove, further comprises providing labelling means for the one or morebinding partners, suitable labelling means being substantially ashereinbefore described.

The present invention also provides a method of screening forautoantibodies produced in response to a TSH receptor in a sample ofbody fluid obtained from a subject suspected of suffering from,susceptible to, having or recovering from autoimmune disease associatedwith an immune reaction to a TSH receptor, said method comprising:

-   -   (a) providing said sample of body fluid from said subject;    -   (b) contacting said sample with        -   (i) a full length TSH receptor, one or more epitopes thereof            or a polypeptide comprising one or more epitopes of a TSH            receptor, and        -   (ii) one or more binding members for a TSH receptor;        -   (suitably under conditions that allow interaction of a TSH            receptor with autoantibodies produced in response to a TSH            receptor) so as to permit said TSH receptor, said one or            more epitopes thereof or said polypeptide, to interact with            either autoantibodies to a TSH receptor present in said            sample, or said one of more binding members; and    -   (c) monitoring the interaction of said TSH receptor, said one or        more epitopes thereof or said polypeptide, with said        autoantibodies present in said sample, thereby providing an        indication of the presence of said autoantibodies to a TSH        receptor in said sample;        -   wherein said one or more binding members are directly or            indirectly immobilised to a surface either prior to, or            after step (b).

Typically the one or more binding members comprise one or more bindingpartners according to the present invention substantially ashereinbefore described. Suitably, labelling means are provided for theTSH receptor, the one or more epitopes thereof or the polypeptide.

The present invention also provides a kit for screening forautoantibodies to a TSH receptor in a sample of body fluid obtained froma subject suspected of suffering from, susceptible to, having orrecovering from autoimmune disease associated with an immune reaction toa TSH receptor, said kit comprising:

-   -   (a) a full length TSH receptor, one or more epitopes thereof or        a polypeptide comprising one or more epitopes of a TSH receptor;    -   (b) one or more binding partners substantially as hereinbefore        described;    -   (c) means for contacting said sample of body fluid from said        subject, said TSH receptor, said one or more epitopes thereof or        said polypeptide, and said one or more binding partners,        (suitably under conditions that allow interaction of a TSH        receptor with autoantibodies produced in response to a TSH        receptor) so as to permit said TSH receptor, said one or more        epitopes thereof or said polypeptide, to interact with either        autoantibodies to a TSH receptor present in said sample, or said        one or more binding partners; and    -   (d) means for monitoring the interaction of said TSH receptor,        said one or more epitopes thereof or said polypeptide, with said        autoantibodies present in said sample, thereby providing an        indication of the presence of said autoantibodies to a TSH        receptor in said sample.

Suitably, a kit as referred to above further comprises labelling meansfor the one or more binding partners, suitable labelling means beingsubstantially as hereinbefore described.

The present invention also provides a kit for screening forautoantibodies to a TSH receptor in a sample of body fluid obtained froma subject suspected of suffering from, susceptible to, having orrecovering from autoimmune disease associated with an immune reaction toa TSH receptor, said kit comprising:

-   -   (a) a full length TSH receptor, one or more epitopes thereof or        a polypeptide comprising one or more epitopes of a TSH receptor;    -   (b) one or more binding members for a TSH receptor;    -   (c) means for contacting said sample of body fluid from said        subject, said TSH receptor, said one or more epitopes thereof or        said polypeptide, and said one or more binding members,        (suitably under conditions that allow interaction of a TSH        receptor with autoantibodies produced in response to a TSH        receptor) so as to permit said TSH receptor, said one or more        epitopes thereof or said polypeptide, to interact with either        autoantibodies to a TSH receptor present in said sample, or said        one or more binding members;    -   (d) means for directly or indirectly immobilising said one or        more members to a surface, either before or after contacting        said one or more binding members with said sample of body fluid        from said subject and said TSH receptor, said one or more        epitopes thereof or said polypeptide; and    -   (e) means for monitoring the interaction of said TSH receptor,        said one or more epitopes thereof or said polypeptide, with said        autoantibodies present in said sample, thereby providing an        indication of the presence of said autoantibodies to a TSH        receptor in said sample.

Typically the one or more binding members comprise one or more bindingpartners according to the present invention substantially ashereinbefore described. Suitably, labelling means are provided for theTSH receptor, the one or more epitopes thereof or the polypeptide.

Suitably a method or kit as referred to above can employ a polypeptideor epitope according to the present invention substantially ashereinbefore described.

Substantially as hereinbefore described, in the presence ofautoantibodies to the TSH receptor, binding of the TSH receptor to theimmobilised binding member or binding partner will be decreased. Such amethod and kit for screening for autoantibodies to a TSH receptor can beadvantageous in alleviating problems that can be associated with TSHreceptor when immobilised to a surface.

A binding partner substantially as hereinbefore described can also beusefully employed in therapy. There is, therefore, further provided bythe present invention methods of treatment comprising administration ofa specific binding partner substantially as hereinbefore described,pharmaceutical compositions comprising a specific binding partnersubstantially as hereinbefore described (together with one or morepharmaceutically acceptable carriers, diluents or excipients therefor),and use of a specific binding partner substantially as hereinbeforedescribed in the manufacture of a medicament or composition, inparticular a medicament or composition for use in stimulating thyroidtissue, and/or tissue containing a TSH receptor. In particular, aspecific binding partner according to the present invention can beemployed in oncology, and in particular for use in the diagnosis,management and treatment of thyroid cancer.

Pharmaceutical compositions according to the present invention includethose suitable for oral, parenteral and topical administration, althoughthe most suitable route will generally depend upon the condition of apatient and the specific disease being treated. The precise amount of abinding partner substantially as hereinbefore described to beadministered to a patient will be the responsibility of an attendantphysician, although the dose employed will depend upon a number offactors, including the age and sex of the patient, the specific diseasebeing treated and the route of administration substantially as describedabove.

There is further provided by the present invention a method ofstimulating thyroid tissue, and/or tissue containing a TSH receptor,which method comprises administering to a patient in need of suchstimulation a diagnostically or therapeutically effective amount of abinding partner substantially as hereinbefore described.

The present invention also provides in combination, a binding partnersubstantially as hereinbefore described, together with one or morefurther agents capable of stimulating thyroid tissue, and/or tissuecontaining a TSH receptor, for simultaneous, separate or sequential usein stimulating thyroid tissue, and/or tissue containing a TSH receptor.Preferably the one or more further agents comprise recombinant human TSHand/or one or more variants, analogs, derivatives or fragments thereof,or variants, analogs or derivatives of such fragments. Alternatively,the one or more further agents can act independently of binding to theTSH receptor.

The following illustrative explanations are provided to facilitateunderstanding of certain terms used herein. The explanations areprovided as a convenience and are not limitative of the invention

BINDING PARTNER, or BINDING MEMBER, FOR A TSH RECEPTOR, describes amolecule having a binding specificity for a TSH receptor. A bindingpartner or binding member as described herein may be naturally derivedor wholly or partially synthetically produced. Such a binding partner orbinding member has a domain or region which specifically binds to and istherefore complementary to one or more epitope regions of a TSHreceptor.

C DOMAIN denotes a region of relatively constant amino acid sequence inantibody molecules.

CDR denotes complementary determining regions which are present on bothheavy and light chains of antibody molecules and represent regions ofmost sequence variability. CDRs represent approximately 15 to 20% ofvariable domains and represent antigen binding sites of an antibody.

FR denotes framework regions and represent the remainder of the variablelight domains and variable heavy domains not present in CDRs.

HC denotes part of a heavy chain of an antibody molecule comprising theheavy chain variable domain and the first domain of an IgG constantregion.

HOST CELL is a cell which has been transformed or transfected, or iscapable of transformation or transfection by an exogenous polynucleotidesequence.

IDENTITY, as known in the art, is the relationship between two or morepolypeptide sequences, or two or more polynucleotide sequences, asdetermined by comparing the sequences.

LC denotes a light chain of an antibody molecule.

STIMULATION OF A TSH RECEPTOR by a binding partner or binding member asdescribed herein denotes the ability of the binding partner or bindingmember to bind to a TSH receptor and to thereby effect, for example,production of cyclic AMP as a result of such binding to the TSHreceptor. Such stimulation is analogous to the responses seen on bindingof TSHE or TSH receptor autoantibodies, to a TSH receptor and in thisway a binding partner or binding member as described herein mimics theeffect of TSH, or TSH receptor autoantibody, binding to a TSH receptor.

V DOMAIN denotes a region of highly variable amino acid sequence inantibody molecules.

V_(H) DOMAIN denotes variable regions or domains in heavy chains ofantibody molecules.

V_(L) DOMAIN denotes variable regions or domains in light chains ofantibody molecules.

The present invention will now be illustrated by the following Figuresand Examples, which do not limit the scope of the invention in any way.

FIG. 1 lists amino acids 1 to 200 of (in the following order) human(HTSHR.PRO; SEQ ID NO:1), porcine (PTSHR.PRO; SEQ ID NO:2), bovine(BTSHR.PRO; SEQ ID NO:3), feline (CTSHR.PRO; SEQ ID NO:4), canine(DTSHR.PRO; SEQ ID NO:5), mouse (MTSHR.PRO; SEQ ID NO:6), rat(RTSHR.PRO; SEQ ID NO:7) and ovine (STSHRP.PRO; SEQ ID NO:8) TSHreceptors. Majority (SEQ ID NO: 93) represents the consensus sequence.

FIG. 2 lists nucleotide bases 1 to 300 coding for regions of (in thefollowing order) feline (CAT. SEQ; SEQ ID NO:9), bovine (COW. SEQ; SEQID NO:10), canine (DOG. SEQ; SEQ ID NO:11), mouse (MOUSE. SEQ; SEQ IDNO:12), porcine (PTSHR. SEQ; SEQ ID NO:13), rat (RAT. SEQ; SEQ IDNO:14), ovine (SHEEP. SEQ; SEQ ID NO:15) and human (HTSHR. SEQ; SEQ IDNO:16) TSH receptors. Majority (SEQ ID NO: 94) represents the consensussequence.

FIG. 3 lists amino acids 200 to 300 of (in the following order) human(HTSHR.PRO; SEQ ID NO:17), porcine (PTSHR.PRO; SEQ ID NO:18), bovine(BTSHR.PRO; SEQ ID NO:19), feline (CTSHR.PRO; SEQ ID NO:20), canine(DTSHR.PRO; SEQ ID NO:21), mouse (MTSHR.PRO; SEQ ID NO:22), rat(RTSHR.PRO; SEQ ID NO:23) and ovine (STSHRP.PRO; SEQ ID NO:24) TSHreceptors. Majority (SEQ ID NO: 95) represents the consensus sequence.

FIG. 4 lists nucleotide bases 700 to 899 coding for regions of (in thefollowing order) feline (CAT. SEQ; SEQ ID NO:25), bovine (COW. SEQ; SEQID NO:26), canine (DOG. SEQ; SEQ ID NO:27), mouse (MOUSE. SEQ; SEQ IDNO:28), porcine (PTSHR. SEQ; SEQ ID NO:29), rat (RAT. SEQ; SEQ IDNO:30), ovine (SHEEP. SEQ; SEQ ID NO:31) and human (HTSHR. SEQ; SEQ IDNO:32) TSH receptors. Majority (SEQ ID NO: 96) represents the consensussequence.

FIG. 5 lists amino acids 250 to 449 of (in the following order) human(HTSHR.PRO; SEQ ID NO:33), porcine (PTSHR.PRO; SEQ ID NO:34), bovine(BTSHR.PRO; SEQ ID NO:35), feline (CTSHR.PRO; SEQ ID NO:36), canine(DTSHR.PRO; SEQ ID NO:37), mouse (MTSHR.PRO; SEQ ID NO:38), rat(RTSHR.PRO; SEQ ID NO:39) and ovine (STSHRP.PRO; SEQ ID NO:40) TSHreceptors. Majority (SEQ ID NO: 97) represents the consensus sequence.

FIG. 6 lists nucleotide bases 750 to 1100 coding for regions of (in thefollowing order) feline (CAT. SEQ; SEQ ID NO:41), bovine (COW. SEQ; SEQID NO:42), canine (DOG. SEQ; SEQ ID NO:43), mouse (MOUSE. SEQ; SEQ IDNO:44), porcine (PTSHR. SEQ; SEQ ID NO:45), rat (RAT. SEQ; SEQ IDNO:46), ovine (SHEEP. SEQ; SEQ ID NO:47) and human (HTSHR. SEQ; SEQ IDNO:48) TSH receptors. Majority (SEQ ID NO: 98) represents the consensussequence.

FIG. 7 lists amino acids 350 to 500 of (in the following order) human(HTSHR.PRO; SEQ ID NO:49), porcine (PTSHR.PRO; SEQ ID NO:50), bovine(BTSHR.PRO; SEQ ID NO:51), feline (CTSHR.PRO; SEQ ID NO:52), canine(DTSHR.PRO; SEQ ID NO:53), mouse (MTSHR.PRO; SEQ ID NO:54), rat(RTSHR.PRO; SEQ ID NO:55) and ovine (STSHRP.PRO; SEQ ID NO:56) TSHreceptors. Majority (SEQ ID NO: 99) represents the consensus sequence.

FIG. 8 lists nucleotide bases 1100 to 1299 coding for regions of (in thefollowing order) feline (CAT. SEQ; SEQ ID NO:57), bovine (COW. SEQ; SEQID NO:58), canine (DOG. SEQ; SEQ ID NO:59), mouse (MOUSE. SEQ; SEQ IDNO:60), porcine (PTSHR. SEQ; SEQ ID NO:61), rat (RAT. SEQ; SEQ IDNO:62), ovine (SHEEP. SEQ; SEQ ID NO:63) and human (HTSHR. SEQ; SEQ IDNO:64) TSH receptors. Majority (SEQ ID NO: 100) represents the consensussequence.

FIG. 9 lists amino acids of the heavy chain (HC) of 4D7 (SEQ ID NO:65).

FIG. 10 lists amino acids of the heavy chain (HC) of 4D7 (SEQ ID NO:65),showing the variable region or domain (namely amino acid numbers 10 to115), the CDRs (namely CDR1 amino acid numbers 31 to 35, CDRII aminoacid numbers 50 to 66 and CDRIII amino acid numbers 99 to 104) and theconstant region or domain (namely amino acid numbers 116 to 200).

FIG. 11 lists amino acids of the light chain (LC) of 4D7 (SEQ ID NO:66).

FIG. 12 lists amino acids of the light chain (LC) of 4D7 (SEQ ID NO:66),showing the variable region or domain (namely amino acid numbers 9 to111), the CDRs (namely CDR1 amino acid numbers 24 to 38, CDRII aminoacid numbers 54 to 60 and CDRIII amino acid numbers 93 to 101) and theconstant region or domain (namely amino acids numbers 112 to 211).

FIG. 13 lists amino acids of the heavy chain (HC) of 16E5 (SEQ IDNO:67).

FIG. 14 lists amino acids of the heavy chain (HC) of 16E5 (SEQ IDNO:67), showing the variable region or domain (namely amino acid numbers9 to 120), the CDRs (namely CDR1 amino acid numbers 31 to 35, CDRIIamino acid numbers 50 to 66 and CDRIII amino acid numbers 99 to 109) andthe constant region or domain (namely amino acid numbers 121 to 205).

FIG. 15 lists amino acids of the light chain (LC) of 16E5 (SEQ IDNO:68).

FIG. 16 lists amino acids of the light chain (LC) of 16E5 (SEQ IDNO:68), showing the variable region or domain (namely amino acid numbers9 to 107), the CDRs (namely CDR1 amino acid numbers 24 to 34, CDRIIamino acid numbers 50 to 56 and CDRIII amino acid numbers 89 to 97) andthe constant region or domain (namely amino acids numbers 108 to 207).

FIG. 17 lists amino acids of the heavy chain (HC) of 17D2 (SEQ IDNO:69).

FIG. 18 lists amino acids of the heavy chain (HC) of 17D2 (SEQ IDNO:69), showing the variable region or domain (namely amino acid numbers9 to 120), the CDRs (namely CDR1 amino acid numbers 31 to 35, CDRIIamino acid numbers 50 to 66 and CDRIII amino acid numbers 99 to 109) andthe constant region or domain (namely amino acid numbers 121 to 205).

FIG. 19 lists amino acids of the light chain (LC) of 17D2 (SEQ IDNO:70).

FIG. 20 lists amino acids of the light chain (LC) of 17D2 (SEQ IDNO:70), showing the variable region or domain (namely amino acid numbers9 to 107), the CDRs (namely CDR1 amino acid numbers 24 to 34, CDRIIamino acid numbers 50 to 56 and CDRIII amino acid numbers 89 to 97) andthe constant region or domain (namely amino acids numbers 108 to 207).

FIG. 21 lists amino acids of the heavy chain (HC) of 14D3 (SEQ IDNO:71).

FIG. 22 lists amino acids of the heavy chain (HC) of 14D3 (SEQ IDNO:71), showing the variable region or domain (namely amino acid numbers9 to 120), the CDRs (namely CDR1 amino acid numbers 31 to 35, CDRIIamino acid numbers 50 to 66 and CDRIII amino acid numbers 99 to 109) andthe constant region or domain (namely amino acid numbers 121 to 205).

FIG. 23 lists amino acids of the light chain (LC) of 14D3 (SEQ IDNO:72).

FIG. 24 lists amino acids of the light chain (LC) of 14D3 (SEQ IDNO:72), showing the variable region or domain (namely amino acid numbers9 to 107), the CDRs (namely CDR1 amino acid numbers 24 to 34, CDRIIamino acid numbers 50 to 56 and CDRIII amino acid numbers 89 to 97) andthe constant region or domain (namely amino acids numbers 108 to 207).

FIG. 25 lists nucleotide bases encoding amino acids of the heavy chain(HC) of 4D7 (SEQ ID NO:73) as shown in FIG. 9.

FIG. 26 lists nucleotide bases encoding amino acids of the heavy chain(HC) of 4D7 (SEQ ID NO:73) as shown in FIG. 9, and shows the nucleotidebases encoding the variable region or domain, the CDRs and the constantregion or domain as shown in FIG. 10.

FIG. 27 lists nucleotide bases encoding amino acids of the light chain(LC) of 4D7 (SEQ ID NO:74) as shown in FIG. 11.

FIG. 28 lists nucleotide bases encoding amino acids of the light chain(LC) of 4D7 (SEQ ID NO:74) as shown in FIG. 11, and shows the nucleotidebases encoding the variable region or domain, the CDRs and the constantregion or domain as shown in FIG. 12.

FIG. 29 lists nucleotide bases encoding amino acids of the heavy chain(HC) of 16E5 (SEQ ID NO:75) as shown in FIG. 13.

FIG. 30 lists nucleotide bases encoding amino acids of the heavy chain(HC) of 16E5 (SEQ ID NO:75) as shown in FIG. 13, and shows thenucleotide bases encoding the variable region or domain, the CDRs andthe constant region or domain as shown in FIG. 14.

FIG. 31 lists nucleotide bases encoding amino acids of the light chain(LC) of 16E5 (SEQ ID NO:76) as shown in FIG. 15.

FIG. 32 lists nucleotide bases encoding amino acids of the light chain(LC) of 16E5 (SEQ ID NO:76) as shown in FIG. 15, and shows thenucleotide bases encoding the variable region or domain, the CDRs andthe constant region or domain as shown in FIG. 16.

FIG. 33 lists nucleotide bases encoding amino acids of the heavy chain(HC) of 17D2 (SEQ ID NO:77) as shown in FIG. 17.

FIG. 34 lists nucleotide bases encoding amino acids of the heavy chain(HC) of 17D2 (SEQ ID NO:77) as shown in FIG. 17, and shows thenucleotide bases encoding the variable region or domain, the CDRs andthe constant region or domain as shown in FIG. 18.

FIG. 35 lists nucleotide bases encoding amino acids of the light chain(LC) of 17D2 (SEQ ID NO:78) as shown in FIG. 19.

FIG. 36 lists nucleotide bases encoding amino acids of the light chain(LC) of 17D2 (SEQ ID NO:78) as shown in FIG. 19, and shows thenucleotide bases encoding the variable region or domain, the CDRs andthe constant region or domain as shown in FIG. 20.

FIG. 37 lists nucleotide bases encoding amino acids of the heavy chain(HC) of 14D3 (SEQ ID NO:79) as shown in FIG. 21.

FIG. 38 lists nucleotide bases encoding amino acids of the heavy chain(HC) of 14D3 (SEQ ID NO:79) as shown in FIG. 21, and shows thenucleotide bases encoding the variable region or domain, the CDRs andthe constant region or domain as shown in FIG. 22.

FIG. 39 lists nucleotide bases encoding amino acids of the light chain(LC) of 14D3 (SEQ ID NO:80) as shown in FIG. 23.

FIG. 40 lists nucleotide bases encoding amino acids of the light chain(LC) of 14D3 (SEQ ID NO:80) as shown in FIG. 23, and shows thenucleotide bases encoding the variable region or domain, the CDRs andthe constant region or domain as shown in FIG. 24.

FIG. 41 lists amino acids of the heavy chain (HC) of 3B3 (SEQ ID NO:81).

FIG. 42 lists amino acids of the heavy chain (HC) of 3B3 (SEQ ID NO:81),showing the variable region or domain (namely amino acid numbers 8 to112), the CDRs (namely CDR1 amino acid numbers 31 to 35, CDRII aminoacid numbers 50 to 66 and CDRIII amino acid numbers 99 to 101) and theconstant region or domain (namely amino acid numbers 113 to 196).

FIG. 43 lists amino acids of the light chain (LC) of 3B3 (SEQ ID NO:82).

FIG. 44 lists amino acids of the light chain (LC) of 3B3 (SEQ ID NO:82),showing the variable region or domain (namely amino acid numbers 9 to111), the CDRs (namely CDR1 amino acid numbers 24 to 38, CDRII aminoacid numbers 54 to 60 and CDRIII amino acid numbers 93 to 101) and theconstant region or domain (namely amino acids numbers 112 to 211).

FIG. 45 lists amino acids of the heavy chain (HC) of 3C7 (SEQ ID NO:83).

FIG. 46 lists amino acids of the heavy chain (HC) of 3C7 (SEQ ID NO:83),showing the variable region or domain (namely amino acid numbers 10 to115), the CDRs (namely CDR1 amino acid numbers 31 to 35, CDRII aminoacid numbers 50 to 66 and CDRIII amino acid numbers 99 to 104) and theconstant region or domain (namely amino acid numbers 116 to 200).

FIG. 47 lists amino acids of the light chain (LC) of 3C7 (SEQ ID NO:84).

FIG. 48 lists amino acids of the light chain (LC) of 3C7 (SEQ ID NO:84),showing the variable region or domain (namely amino acid numbers 9 to111), the CDRs (namely CDR1 amino acid numbers 24 to 38, CDRII aminoacid numbers 54 to 60 and CDRIII amino acid numbers 93 to 101) and theconstant region or domain (namely amino acids numbers 112 to 211).

FIG. 49 lists amino acids of the heavy chain (HC) of 2B4 (SEQ ID NO:85).FIG. 50 lists amino acids of the heavy chain (HC) of 2B4 (SEQ ID NO:85),showing the variable region or domain (namely amino acid numbers 9 to122), the CDRs (namely CDR1 amino acid numbers 31 to 35, CDRII aminoacid numbers 50 to 66 and CDRIII amino acid numbers 99 to 111) and theconstant region or domain (namely amino acid numbers 123 to 207).

FIG. 51 lists amino acids of the light chain (LC) of 2B4 (SEQ ID NO:86).

FIG. 52 lists amino acids of the light chain (LC) of 2B4 (SEQ ID NO:86),showing the variable region or domain (namely amino acid numbers 9 to112), the CDRs (namely CDR1 amino acid numbers 24 to 39, CDRII aminoacid numbers 78 to 82 and CDRIII amino acid numbers 94 to 102) and theconstant region or domain (namely amino acids numbers 113 to 212).

FIG. 53 lists nucleotide bases encoding amino acids of the heavy chain(HC) of 3B3 (SEQ ID NO:87) as shown in FIG. 41.

FIG. 54 lists nucleotide bases encoding amino acids of the heavy chain(HC) of 3B3 (SEQ ID NO:87) as shown in FIG. 41, and shows the nucleotidebases encoding the variable region or domain, the CDRs and the constantregion or domain as shown in FIG. 42.

FIG. 55 lists nucleotide bases encoding amino acids of the light chain(LC) of 3B3 (SEQ ID NO:88) as shown in FIG. 43.

FIG. 56 lists nucleotide bases encoding amino acids of the light chain(LC) of 3B3 (SEQ ID NO:88) as shown in FIG. 43, and shows the nucleotidebases encoding the variable region or domain, the CDRs and the constantregion or domain as shown in FIG. 44.

FIG. 57 lists nucleotide bases encoding amino acids of the heavy chain(HC) of 3C7 (SEQ ID NO:89) as shown in FIG. 45.

FIG. 58 lists nucleotide bases encoding amino acids of the heavy chain(HC) of 3C7 (SEQ ID NO:89) as shown in FIG. 45, and shows the nucleotidebases encoding the variable region or domain, the CDRs and the constantregion or domain as shown in FIG. 46.

FIG. 59 lists nucleotide bases encoding amino acids of the light chain(LC) of 3C7 (SEQ ID NO:90) as shown in FIG. 47.

FIG. 60 lists nucleotide bases encoding amino acids of the light chain(LC) of 3C7 (SEQ ID NO:90) as shown in FIG. 47, and shows the nucleotidebases encoding the variable region or domain, the CDRs and the constantregion or domain as shown in FIG. 48.

FIG. 61 lists nucleotide bases encoding amino acids of the heavy chain(HC) of 2B4 (SEQ ID NO:91) as shown in FIG. 49.

FIG. 62 lists nucleotide bases encoding amino acids of the heavy chain(HC) of 2B4 (SEQ ID NO:91) as shown in FIG. 49, and shows the nucleotidebases encoding the variable region or domain, the CDRs and the constantregion or domain as shown in FIG. 50.

FIG. 63 lists nucleotide bases encoding amino acids of the light chain(LC) of 2B4 (SEQ ID NO:92) as shown in FIG. 51.

FIG. 64 lists nucleotide bases encoding amino acids of the light chain(LC) of 2B4 (SEQ ID NO:92) as shown in FIG. 51, and shows the nucleotidebases encoding the variable region or domain, the CDRs and the constantregion or domain as shown in FIG. 52.

More specifically, the FIGS. 1 to 8 illustrate the following:

FIG. 1 lists amino acids 1 to 200 of TSH receptors in the abovementioned species, which include the following amino acid sequencesemployed in the present invention:

-   -   amino acids 22 to 91 of a TSH receptor,    -   amino acids 32 to 41 of a TSH receptor, and    -   amino acids 36 to 42 of a TSH receptor.

FIG. 2 lists nucleotide bases 1 to 300 in the above mentioned species,which include coding regions for the above mentioned amino acidsequences present in FIG. 1.

FIG. 3 lists amino acids 200 to 300 of TSH receptors in the abovementioned species, which include the following amino acid sequencesemployed in the present invention:

-   -   amino acids 246 to 260 of a TSH receptor, and    -   amino acids 247 to 260 of a TSH receptor.

FIG. 4 lists nucleotide bases 700 to 899 in the above mentioned species,which include coding regions for the above mentioned amino acidsequences present in FIG. 3.

FIG. 5 lists amino acids 250 to 449 of TSH receptors in the abovementioned species, which include the following amino acid sequencesemployed in the present invention:

-   -   amino acids 260 to 363 of a TSH receptor; and    -   amino acids 277 to 296 of a TSH receptor.

FIG. 6 lists nucleotide bases 750 to 1100 in the above mentionedspecies, which include coding regions for the above mentioned amino acidsequences present in FIG. 5.

FIG. 7 lists amino acids 350 to 500 of TSH receptors in the abovementioned species, which include the following amino acid sequencesemployed in the present invention:

-   -   amino acids 380 to 418 of a TSH receptor; and    -   amino acids 381 to 385 of a TSH receptor.

FIG. 8 lists nucleotide bases 1100 to 1299 in the above mentionedspecies, which include coding regions for the above mentioned amino acidsequences present in FIG. 7.

EXAMPLE 1 (1) Production of Mouse Monoclonal Antibodies to the TSHReceptor

BALB/c mice were immunised with a recombinant, highly purified matureform of the TSH receptor expressed in CHO cells. [Y Oda, J Sanders, MEvans, A Kiddie, A Munkley, C James, T Richards, J Wills, J Furmaniak, BRees Smith “Epitope analysis of the human thyrotrophin (TSH) receptorusing monoclonal antibodies.” Thyroid 2000 10(12): 1051-1059.] Mouseantibodies were also raised by DNA immunization technique with fulllength human TSHR cDNA cloned in pcDNA3.1. MAbs were cloned usingstandard techniques and IgGs were purified from culture supernatants byaffinity chromatography on Protein A Sepharose. The reactivity of MAbswith the TSH receptor was tested by (a) Western blotting with partiallypurified receptors, (b) inhibition of TSH binding to the TSH receptor,and (c) immunoprecipitation of ³⁵S-labelled TSH receptors produced in anin vitro transcription/translation system as described in Y Oda, JSanders, S Roberts, M Maruyama, R Kato, M Perez, V B Peteresen, NWedlock, J Furmaniak, B Rees Smith “Binding characteristics ofantibodies to the TSH receptor.” Journal of Molecular Endocrinology 199820: 233-244.

(2) Inhibition of ¹²⁵I-TSH Binding to the TSH Receptor

The inhibition of ¹²⁵I-TSH binding to the TSH receptor was analysed inan assay where, 50 μL of detergent solubilised TSH receptor waspreincubated with 50 μL of MAb purified as described in step (1) for 15minutes at room temperature before addition of 100 μL of ¹²⁵I-TSH(30,000 cpm) followed by incubation at 37° C. for one hour. Thecomplexes of ¹²⁵I-TSH/TSH receptor were precipitated by addition of 2 mL16.5% polyethylene glycol and 25 μL healthy blood donor serum,centrifuged at 1500×g for 30 minutes at 4° C., aspirated and theradioactivity of the pellets counted using known techniques.

MAbs termed: 2B4 MAb (at IgG concentration of 5 μg/mL), 8E2 Mab (at IgGconcentration of 1 ug/mL) and 18C5 Mab (at IgG concentration 1 mg/mL)showed 76%, 38% and 91% inhibition of TSH binding, respectively. Fabfragments were produced from 2B4 Mab, 8E2 Mab and 18C5 Mab IgGs bydigestion with L-cysteine/papain or pepsin, followed by the separationof Fc and Fab on Protein A column.

(3) Epitope Recognition by MAbs

Western blotting analysis [Y Oda, J Sanders, M Evans, A Kiddie, AMunkley, C James, T Richards, J Wills, J Furmaniak, B Rees Smith“Epitope analysis of the human thyrotrophin (TSH) rector usingmonoclonal antibodies.” Thyroid 2000 10(12): 1051-1059.] showed that 2B4MAb bound to an epitope between amino acid (aa) 380 and 418, 8E2 MAb toan epitope between aa 22 and 91 and 18C5 MAb to an epitope between aa246 and 260 of the TSH receptor sequence. Analysis with overlapping TSHreceptor peptides covering these regions [Y Oda, J Sanders, M Evans, AKiddie, A Munkley, C James, T Richards, J Wills, J Furmaniak, B ReesSmith “Epitope analysis of the human thyrotrophin (TSH) receptor usingmonoclonal antibodies.” Thyroid 2000 10(12): 1051-1059.] showed that 2B4MAb reacted with the aa 381 to 385, 8E2 MAb with the aa 36 to 42 and18C5 MAb with the aa 247 to 260.

(4) Preparation of ¹²⁵I-Labelled TSH Receptor

Solubilised preparations of TSH receptor were labelled with ¹²⁵I by wayof ¹²⁵I-labelled MAB (4E31) reactive with the C-terminal end of the TSHreceptor prepared as described in J Sanders, Y Oda, S Roberts, A Kiddie,T Richards, J Bolton, V McGrath, S Walters, D Jaskolski, J Furmaniak, BRees Smith “The interaction of TSH receptor autoantibodies with¹²⁵I-labelled TSH receptor.” Journal of Clinical Endocrinology andMetabolism 1999 84(10):3797-3802. Aliquots of ¹²⁵I-labelled 4E31 F(ab)₂were incubated for 15 minutes at room temperature with solubilised TSHreceptor and then used an immunoprecipitation assay as described in step(5).

(5) Inhibition of TSH Receptor Autoantibody (TRAb) Binding to the TSHReceptor by MAbs

The inhibition of TRAb binding to the TSH receptor by MAbs was tested asfollows:

10 μL of ¹²⁵I-labelled TSH receptor (30,000 cpm) prepared in step (4)was preincubated with 20 μL of 2B4 Fab (5 and 10 mg/mL) for 15 minutesat room temperature followed by incubation with 20 μL of TRAb positivepatient serum for one hour at room temperature. 50 μL of solid phaseProtein A (an anti-human IgG reagent) was then added and incubationcontinued for one hour at room temperature followed by washing step andcentrifugation at 1500×g at 4° C. for 30 minutes; aspiration andcounting of the radioactivity of the pellets. Similar experiments werecarried out with 8E2 and 18C5 Fabs and the combination of two Fabstogether.

Results of Example 1

Results of the inhibition of TRAb binding to the TSH receptor are shownin Table 1.

EXAMPLE 2 Methods (1) Production of Mouse Monoclonal Antibodies to theTSH Receptor

BALB/C mice were immunised with a recombi-nant, highly purified matureform of the TSH receptor expressed in CHO cells (Y. Oda, J. Sanders, M.Evans, A. Kiddie, A. Munkley, C. James, T. Richards, J. Wills, J.Furmaniak, B. Rees Smith “Epitope analysis of the human thyrotropin(TSH) receptor using monoclonal antibodies” Thyroid 2000 10(12):1051-1059). Mouse antibodies were also raised by DNA immunisationtechniques with full length human TSHR cDNA cloned in pRC/CM.1. MAbswere cloned using standard techniques and IgGs were purified fromculture supernatants by affinity chromatography on Protein A Sepharose.

(Fab)₂ fragments were produced from the purified MAb IgGs by digestionwith pepsin followed by chromatography on a protein A affinity column asdescribed in Y. Oda, J. Sanders, S. Roberts, M. Maruyama, R P Kato, M.Perez, V B Petersen, N. Wedlock, J. Furmaniak, B. Rees Smith 1998“Binding characteristics of antibodies to the TSH receptor”. Journal ofMolecular Endocrinology 20: 233-244.

Fab fragments were prepared by digestion of the purified MAbs withpapain as described in E. Hendry, G. Taylor, F. Grennan-Jones, A.Sullivan, N. Liddy, J. Godfrey, N. Hayakawa, M. Powell J. Furmaniak, BRees Smith 2001 “X-ray crystal structure of a monoclonal antibody thatbinds to a major autoantigenic epitope on thyroid peroxidase.” Thyroid11(12): 1091-1099.

The reactivity of MAbs with the TSH receptor was tested by (a) westernblotting with partially purified receptors, (b) inhibition of the TSHbinding to the TSH receptor and (c) immunoprecipitation of ³⁵S-labelledTSH receptors produced in an in vitro transcription/translation systemas described in Y. Oda, J. Sanders, S. Roberts, M. Maruyama, R. Kato, M.Perez, V B. Petersen, N. Wedlock, J. Furmaniak, B. Rees Smith “Bindingcharacteristics of antibodies to the TSH receptor” Journal of MolecularEndocrinology 1998 20: 233-244.

(2) Inhibition of ¹²⁵I TSH Binding to the TSH Receptor

(a) PEG Method for Use with Detergent Solubilised TSHR

The inhibition of ¹²⁵I TSH binding to detergent solubilised TSH receptorwas analysed in an assay where 50 μL of MAb purified as described inMethods (1) above was preincubated with receptor for 15 minutes at roomtemperature before addition of 100 μL of ¹²⁵I TSH (30,000 cpm) followedby incubation at 37° C. for one hour. The complexes of ¹²⁵I TSH and TSHreceptor were precipitated by addition of 2 mL 16.5% polyethylene glycoland 25 μL healthy blood donor serum, centrifuged at 1500×g for 30minutes at 4° C., aspirated and the radioactivity of the pellets countedin a gamma counter.

(b) Method Using Tubes Coated with TSHR

In this procedure, plastic tubes are first coated with a MAb such as4E31 which binds to a part of the TSHR unrelated to TSH or TRAb binding.Detergent solubilised TSHR preparations are then added, captured by theTSHR MAb and then become immobilised on the tube surface in such a wayas to be able to bind TSH or TRAb. In particular the MAb 4E31 reactivewith the TSHR C terminus (10 μg/mL F(ab)₂ preparation in 0.1 M Na₂CO₃ pH9.2) was added to plastic tubes (Nune Maxisorp, 200 μL per tube) andcoating allowed to proceed overnight 4° C. After washing andpost-coating (10 mg/mL bovine serum albumin) the tubes were washed againwith assay buffer (10 mM Tris-HCl pH 7.8, 50 mM NaCl, 1 mg/mL bovineserum albumin, 0.1% Triton X-100). 200 μL of a detergent solubilisedTSHR preparation was then added and incubated overnight at 4° C.followed by aspiration and washing steps. Thereafter, 20 μL of “start”buffer (10 mM Tris-HCl pH 7.8, 50 mM NaCl, bovine serum albumin 1 mg/mL,6 mM NaN₃, 1% Triton X-100) was added to the TSHR coated tubes followedby 100 μL of purified MAb IgG or patient sera and incubated at roomtemperature for 2 hours with gentle shaking. After aspiration, the tubeswere washed twice with 1 mL of assay buffer before addition of 100 μL of¹²⁵I TSH (80,000 cpm) and incubation at room temperature for 20-60 minwith shaking. The tubes were then washed twice with 1 mL of assaybuffer, aspirated and counted in a gamma counter.

(3) Analysis of Thyroid Stimulating or Blocking Activities of MAbs.

The ability of MAbs to either stimulate the production of cyclic AMP inisolated porcine thyroid cells (thyroid stimulating activity) or to actas TSH antagonists by blocking TSH stimulation of cyclic AMP (blockingactivity) was assessed using reagents from Yamasa Corporation, Tokyo,Japan.

In addition the ability of the MAbs to stimulate production of cyclicAMP in Chinese hamster ovary (CHO) cells expressing human TSHR wasanalysed as described by M. Kita, L. Ahma, P C. Marians, H. Viase, P.Unger, P. N. Graves, T. F. Davies 1999 “Regulation and transfer of amurine model of thyrotropin receptor antibody mediated Graves' disease.”Endocrinology 140: 1392-1398.

(4) Binding of ¹²⁵I-Labelled MAbs to the TSHR and Effect of TRAb

Purified IgG from two of the MAbs that showed thyroid simulatingactivity (16E5 and 14D3, table 2) were labelled with ¹²⁵I followed byseparation of unincorporated ¹²⁵I by filtration on Sephadex G-50 as in(4) in Example 1.

Plastic tubes were coated with TSHR preparations as in 2b above.Thereafter, 100 μL of test serum (from healthy blood donors or frompatients with Graves' disease) were added and tubes incubated for 2hours at room temperature with shaking. After this incubation, the tubeswere washed 2 times with assay buffer. Then, 100 μL of ¹²⁵I-labelled16E5 or 14D3 IgG (30,000 cpm diluted in 20 mM Tris-HCl pH 7.3, 50 mMNaCl, 1 mg/mL bovine serum albumin, 0.1% Triton X-100) was added to thetubes and incubated for 1 hour at room temperature with shaking. Thetubes were then washed twice with the same buffer that was used fordiluting ¹²⁵I-labelled MAbs and counted in a gamma counter.

(5) Binding of TSHR to MAb Coated Tubes and Effect of TRAb

Detergent solubilised TSHR preparations (20 μL were incubated for 1 hourat room temperature with 100 μL of test serum and 20 μL of start buffer(2b above). 100 μL of this mixture was then added to plastic tubescoated with TSHR MAb (as in 2b above) and incubated for 1 hour withshaking at room temperature. Then the tubes were aspirated and washedtwice (2b above) and 100 μL (30,000 cpm) of ¹²⁵I-labelled C-terminalTSHR MAb 4E31 F(ab)₂ preparation labelled with ¹²⁵I as in 4 above added.After further incubation for 1 hour at room temperature with shaking,the tubes were aspirated, washed twice and the radioactivity countedwith a gamma counter.

Oligonucleotide primers were designed using the sequences as describedpreviously (Kettleborough C. A. et al “Optimization of primers forcloning libraries of mouse immunoglobulin genes using the polymerasechain reaction.” European Journal of Immunology 1993 23:206-211).

Both sense and antisense primers included additional 5′ restrictionendonuclease site sequences to facilitate cloning of PCR products.RT-PCR products were cloned into pUC18 DNA prepared by the Qiagen method(Qiagen) and sequenced by the Sanger-Coulson method

Results of Example 2

-   (1) Thyroid stimulating activity of the TSHR MAbs is shown in tables    2 and 3. Four of the MAbs (16E5, 14D3, 17D2, and 4D7) were able to    stimulate cyclic AMP production in isolated porcine thyroid cells.    In addition when Fab fragments from three of these MAbs were tested,    all three also stimulated cyclic AMP production (table 2). For    comparison a TRAb positive patient serum showed similar levels of    stimulation to the MAbs (table 2). Also, TSHR MAb 2B4 which has the    ability to inhibit TSH binding to the TSHR strongly did not show    thyroid stimulating activity (table 2). Another TSHR MAb and Fab    (3B3) did not stimulate cyclic AMP production nor did the Tg MAb Fab    2G2 (table 2).    -   In a further series of experiments some of the MABS which were        able to stimulate porcine thyroid cells (16E5 and 14D3) were        tested for their ability to stimulate cyclic AMP production in        CHO cells expressing human TSHR (table 3). Similar results were        obtained to those observed with porcine thyroid cells.-   (2) In the presence of sera from healthy blood donors, ¹²⁵I-labelled    16E5 bound to TSHR coated tubes is in the range from 23 to 35% of    total counts added (table 4). In the presence of sera from patients    with Graves' disease (all TRAb positive) the binding of    ¹²⁵I-labelled 16E5 was markedly reduced and was in the range from    1.9 to 7.5% (table 4).    -   This indicated that Graves' disease patient sera with TRAb        activity inhibit the binding of TSHR MAb 16E5 to the TSHR.        Further experiments with labelled 16E5 are shown in table 5        where a comparison of the effects of Graves' disease patient        sera on (a) ¹²⁵I-labelled 16E5 to TSHR coated tubes and (b)        ¹²⁵I-labelled TSH binding to TSHR coated tubes. Similar        experiments to those shown in table 5 were carried out with        ¹²⁵I-labelled TSHR MAb 14D3 and the results are shown in table        6.    -   The effects of Graves' disease patient sera on TSHR coated tube        binding by ¹²⁵I-labelled 16E5, 14D3 or TSH were similar with        strong inhibition of binding being observed in most cases        (tables 5 and 6). In contrast to Graves' disease patient sera,        sera from healthy blood donors had little effect on labelled MAb        or labelled TSH binding to TSHR coated tubes (tables 5 and 6).        Table 7 shows the effect of sera containing autoantibodies other        than the TSHR autoantibodies on TSHR coated tube binding by        labelled TSH, 16E5 and 14D3. As can be seen from table 7, sera        containing autoantibodies to glutamic acid decarboxylase (D1 and        D2) or to 21-hydroxylase (A1 and A2) had no effect on TSH or MAb        binding. However, the serum G42 from a patient with Graves'        disease showed a strong, dose-dependent inhibition of both TSH        and MAb binding.-   (3) As shown in table 8, plastic tubes coated with MAb 16E5 were    able to bind TSHR and this binding was inhibited by Graves' sera    containing TSHR autoantibodies. In particular, detection of TSHR    binding by the ¹²⁵I-labelled TSHR MAb 4E31 showed that (a) in the    presence of sera from healthy blood donors, labelled 4E31 binding    ranged from 13.5-17.8% of total cpm added whereas (b) in the    presence of Graves' sera, labelled MAb binding ranged from 1.8-4.8%    of total cpm added. Similar results were obtained with plastic tubes    coated with MAb 14D3 (table 9).

Conclusions

The results shown in tables 2-9 show:

-   -   (a) we have produced TSHR MAbs and MAb Fab fragments which can        stimulate isolated thyroid cells in a similar way to TRAb in        patient sera and in a similar way to TSH. Different MAbs show        different degrees of stimulating activity.    -   (b) these MAbs can be used instead of labelled TSH in assays for        TSHR autoantibodies (TRAb).    -   (c) when the MAbs are coated onto plastic surfaces, they can        bind TSHR preparations. This binding is inhibited by TRAb in        patient sera, thus providing a new type of TRAb assay.    -   (d) the ability of the MAbs to stimulate the thyroid means that        they are potentially useful as alternatives to TSH in in vivo        applications.

EXAMPLE 3 Inhibition of ¹²⁵I-16E5 Fab Binding to Solubilised TSHReceptor by TSH Receptor Mabs Method

The inhibition of 11I-16E5 Fab binding to detergent solubilised TSHreceptor was analysed in an assay where 50 μL of Mab IgG (100 μg/ml)purified as described above was incubated with receptor for 30 minutesat room temperature before addition of 100 μL of ¹²⁵I-16E5 Fab (30,000cpm) followed by incubation at room temperature for 2 hours. Thecomplexes of ¹²⁵I-16E5 Fab and TSH receptor were precipitated byaddition of 2 ml 16.5% polyethylene glycol and 50 μL healthy blood donorserum, centrifuged at 1500×g for 30 minutes at 4° C., aspirated and theradioactivity of the pellets counted in a gamma counter.

The results are shown in Table 10. From Table 10 it can be seen that Mab4D7 (which binds to epitope region 246 to 260 and stimulates isolatedthyroid cells) quite strongly inhibits labelled 16E5 Fab binding to theTSH receptor (24.2% inhibition). Two other Mabs, 3C7 and 18C5 also quitestrongly inhibit 16E5 Fab binding (17 and 15.7% inhibition respectively)and also bind to the epitope region 246 to 260. Weak or no inhibition isobserved with the other Mabs. This suggests that epitope region 246 to260 is involved in 16E5 binding to the TSH receptor. As the otherstimulating Mabs 14D3 and 17D2 compete well with 16E5 binding to the TSHreceptor as can be seen from Table 10, epitope region 246 to 260 isprobably also important for TSH receptor binding by 14D3 and 17D2.

TABLE 1 Inhibition of binding of TRAb in patient serum (K3) to the TSHreceptor by Mab Fabs Serum K3 1/10 Labelled TSHR immunoprecipitated (%)% inhbition Buffer 17.5 — 2B4 (5 mg/mL) 10.1 42 2B4 (10 mg/mL) 3.9 77.718C5 (5 mg/mL) 13.7 21.7 18C5 (10 mg/mL) 9.7 44 8E2 (5 mg/mL) 15.0 14.38E2 (10 mg/mL) 13.0 25.7 2B4 + 18C5 (5 mg/mL) 5.7 67.4 18C5 + 8E2 (5mg/mL) 12.4 29.1 2B4 + 8E2 (5 mg/mL) 8.1 53.7 Unlabelled TSH (2.94mg/mL) 7.8 55.4 2B4 + 8E2 + 18C5 (3.3 mg/mL) 7.4 57.7${\% \mspace{14mu} {inhibition}}\; = {100 - \left( {\frac{A}{B} \times 100} \right)}$where A = ¹²⁵I-TSHR (cpm) immunoprecipitated in the presence of testsera and test Mab Fab as a percentage of total cpm of material added tothe tube; B = ¹²⁵I-TSHR (cpm) immunoprecipitated in the presence of testsera and assay buffer as a percentage of total cpm of material added tothe tube

The above results show that:

-   (1) the sequences of the TSH receptor which are involved in the TSHR    binding are also involved in TRAb binding;-   (2) mouse Mab reactive with these sequences can be used effectively    to inhibit TRAb binding to the TSH receptor; and-   (3) one or more of the MAbs reactive with one or more of the above    TSH receptor sequences can be used to detect and measure TRAb.

TABLE 2 Thyroid stimulating activity of TSHR MAbs tested using isolatedporcine thyroid cells Inhibition of TSH Test sample Stimulation (%)¹binding (%)^(2,3) 16E5 IgG 200 μg/ml 466 nt 20 μg/ml 332 83.3 2 μg/ml269 73.6 0.2 μg/ml 157 nt 0.02 μg/ml 52 nt 14D3 IgG 200 μg/ml 557 nt 20μg/ml 351 76.4 2 μg/ml 323 61.0 0.2 μg/ml 227 nt 0.02 μg/ml 78 nt 17D2IgG 200 μg/ml 377 nt 20 μg/ml 207 81.3 2 μg/ml 134 73.7 4D7 IgG 200μg/ml 259 33⁴ 20 μg/ml 31 nt 3B3 IgG^(a) 200 μg/ml 34 30.7 20 μg/ml 37 6.1 2B4 IgG^(a) 20 μg/ml 100 nt 2 μg/ml 116 69.9 3C7 Fab 1 mg/ml 34845.2 4D7 Fab 1 mg/ml 512 48.6 16E5 Fab 200 μg/ml 425 53⁵ 14D3 Fab 200μg/ml 648 64⁵ 17D2 Fab 200 μg/ml 274 45⁵ 3B3 Fab^(a) 200 μg/ml 42 66.5⁴2G2 Fab⁶ 1 mg/ml 55  0 200 μg/ml 37  0 TRAb +ve patient dil 1:2 771 65⁷dil 1:4 530 nt Pool of healthy blood donor sera 29  0.6 TRAb negativeserum 70  0 Table 2 footnotes ¹MAb IgG or Fab preparations were dilutedin the pool of healthy blood donor sera. Stimulation (%) was calculatedas 100x the ratio of: cyclic AMP produced in the presence of test sampleto cyclic AMP produced in the presence of a pool of healthy blood donorsera. A stimulation level of >180% was assessed as positive i.e. thislevel of stimulation was always greater than that observed by sera fromindividual healthy blood donors. ²Inhibition of TSH binding levelof >10% is positive ³Method = coated tube ⁴Inhibition tested at 250μg/ml ⁵Inhibition tested at 10 μg/ml ⁶2G2 is a MAb reactive withthyroglobulin i.e. unreactive with the TSHR ⁷Inhibition with undilutedserum ^(a)3B3 and 2B4 IgGs act as TSH antagonists i.e. block the abilityof TSH to stimulate cyclic AMP production by isolated porcine thyroidcells. nt = not tested at this concentration

TABLE 3 Inhibition of TSH Test sample¹ Stimulation (%)² binding(%)^(3,4) 16E5 20 μg/mL 850 78.5⁵ 14D3 20 μg/mL 908 71.8⁵ 2B4 20 μg/mL111 84.4 TRAb +ve patient 850 65.0⁶ Pool of healthy blood donor sera 1000 Table 3 footnotes: ¹All samples were diluted 1:10 prior to addition tocells. ²Stimulation (%) was calculated as 100x the ratio of: cyclic AMPproduced in the presence of test sample to cyclic AMP produced in thepresence of a pool of healthy blood donor sera. ³Inhibition of TSHbinding level of >10% is positive. ⁴Method = coated tubes ⁵Inhibitiontested at 10 μg/mL ⁶Tested for inhibition undiluted.

TABLE 4 Binding of ¹²⁵I-labelled Mab 16E5 to TSHR coated tubes andeffect of TRAb in patient sera Inhibition of TSH ¹²⁵I-16E5 bound to TSHRcoated Test Material¹ binding (%)² tubes (% total counts added) G1 215.6 G2 22.7 6.5 G3 24.7 3.5 G4 22.7 6.0 G5 28.1 3.6 G6 29.4 2.5 G7 29.35.8 G8 39 1.9 G9 31.9 6.8 G10 34.8 5.4 G11 34.5 3.4 G12 35.3 4.2 G1335.6 6.2 G14 36.9 2.8 G15 30.3 4.3 G16 35 2.2 G17 47.6 3.9 G18 43 3.4G19 53.5 3.7 G20 59.2 7.5 G21 58.9 4.9 NPS <14 27.5 NSF 1 <14 23.3 NSF 2<14 30.2 NSF 3 <14 29.1 NSF 4 <14 22.8 NSF 5 <14 28.9 NSF 6 <14 31.0 NSF7 <14 29.2 NSF 8 <14 35.3 NSF 9 <14 26.3 NSF 10 <14 25.2 Table 4footnotes: ¹sera G1-G22 are from patients with Graves' disease sera NSF1-NSF 10 are from healthy blood donors NPS = pool of healthy blood donorsera ²Inhibition of TSH binding >14% is positive; PEG method used

TABLE 5 Effect of Graves' disease patient sera on ¹²⁵I-16E5 binding and¹²⁵I-TSH binding to TSHR coated tubes ¹²⁵I-16E5 bound to Inhibition¹²⁵I-TSH bound to TSHR coated tubes of ¹²⁵I-16E5 TSHR coated tubesInhibition of Test (% total counts binding (% total counts ¹²⁵I-TSHmaterial¹ added)² (%)^(2,3) added)⁴ binding (%)^(3,4) G23 13.2 44.0 8.927.1 G28 5.8 75.4 3.8 68.5 G29 13.3 43.6 8.0 34.4 G30 9.2 61.0 5.3 56.9G32 11.9 49.6 7.5 38.4 G36 15.5 34.3 10.1 17.5 G38 16.1 31.8 10.0 18.3G41 17.8 24.6 10.8 11.4 G43 5.9 75.0 4.0 67.2 G44 18.6 21.2 12.4 −ve G455.1 78.4 3.5 71.0 G46 3.8 83.9 2.7 77.9 G47 7.2 69.5 4.3 64.8 G48 6.970.8 4.8 60.8 G49 9.1 61.4 6.1 49.6 G50 8.7 63.1 6.3 48.4 G51 11.9 49.67.9 35.2 G52 12.3 47.9 7.4 39.0 NSF 4 23.0 2.6 12.5 −ve NSF 5 25.3 −ve12.3 −ve NSF 10 22.4 5.1 12.5 −ve NSF 16 23.3 1.3 12.0 1.8 NSF 17 24.2−ve 11.5 5.3 NSF 18 19.9 15.7 11.2 8.0 NSF 20 21.5 8.9 12.3 −ve NSF 2123.3 1.3 12.3 −ve NSF 22 24.5 −ve 12.4 −ve NSF 26 26.5 −ve 12.8 −veTable 5 footnotes: ¹Sera G53-G52 are from patients with Graves' disease;sera NSF are from healthy blood donors ²mean binding in the presence ofhealthy blood donor sera was 23.6% for ¹²⁵I- 16E5 ³inhibition of bindingwas calculated using the formula${\% \mspace{14mu} {inhibition}}\; = {100 - \left( {\frac{A}{B} \times 100} \right)}$where A = binding in the presence of test serum; B = mean binding in thepresence of healthy blood donor sera ⁴mean binding in the presence ofhealthy blood donor sera was 12.2% for ¹²⁵I-TSH

TABLE 6 Binding of ¹²⁵I-labelled MAb 14D3 to TSHR coated tubes andeffect of TRAb in patient sera ¹²⁵I-14D3 bound to Inhibition ¹²⁵I-TSHbound to TSHR coated tubes of ¹²⁵I-14D3 TSHR coated tubes Inhibition ofTest (% total counts binding (% total counts ¹²⁵I-TSH material¹ added)²(%)^(2,3) added)⁴ binding (%)^(3,4) G23 13.9 20 8.9 26.6 G24 11.3 35 6.943.4 G25 14.1 19 7.2 40.5 G26 7.3 58 2.6 78.3 G27 12.3 29.7 7.3 40.1 G288.0 54.4 3.8 68.3 G29 13.2 24.4 8.0 34.0 G30 12.5 28.4 5.3 56.6 G31 9.844 4.3 64.3 G32 11.4 34.8 7.5 38.0 G33 12.7 27.2 6.1 49.9 G34 10.9 37.57.5 37.8 G35 9.8 43.6 4.3 64.6 G36 13.5 22.8 10.1 16.9 G37 11.9 31.6 9.323.4 G38 11.3 35.4 10.0 17.6 G39 12.3 29.5 7.9 34.8 G40 9.8 44.0 7.240.9 G41 14.0 19.8 10.8 10.7 NSF 4 17.4 0.3 12.5 −ve NSF 5 16.5 9.1 12.3−ve NSF 10 17.6 −ve 12.5 −ve NSF 16 17.7 −ve 12.0 1.1 NSF 17 17.0 2.711.5 4.6 NSF 18 16.6 8.6 11.2 7.3 NSF 20 18.3 −ve 12.3 −ve NSF 21 16.83.6 12.3 −ve NSF 22 16.3 6.7 12.4 −ve NSF 26 18.4 −ve 12.8 −ve Table 6footnotes: ¹Sera G23-41 are from patients with Graves' disease Sera NSFare from healthy blood donors ²Mean binding in the presence of healthyblood donor sera was 17.4% for ¹²⁵I- 14D3. ³Inhibition of binding wascalculated using the formula:${\% \mspace{14mu} {inhibition}}\; = {100 - \left( {\frac{A}{B} \times 100} \right)}$where A = binding in the presence of test serum; B = mean binding in thepresence of healthy blood donor sera ⁴Mean binding in the presence ofhealthy blood donor sera was 12.1% for ¹²⁵I- TSH.

TABLE 7 Effect of sera from various patients in TSHR coated tube bindingby labelled TSH, 16E5 and 14D3 % inhibition of binding to TSHR coatedtubes² using: Test sample ¹²⁵I-TSH ¹²⁵I-16E5 ¹²⁵I-14D3 G42/5 87 71 77G42/10 82 56 51 G42/20 70 34 24 D1/10 2 3 2 D1/100 −2 3 0 D2/10 1 1 −7D2/100 −2 0 0 A1/10 −1 3 2 A1/100 −1 3 −1 A2/10 5 3 5 A2/100 1 3 1 Table7 footnotes: ¹Serum G42 is from a patient with Graves' disease; sera D1and D2 are from patients with type 1 diabetes mellitus (positive forauto antibodies to glutamic acid decarboxylase); sera A1 and A2 are frompatients with Addison's disease (positive for steroid 21-hydroxylaseautoantibodies) All test sampes were diluted in a pool of serum fromhealthy blood donors and dilution factor shown as /5, /10, /20 or /100²Inhibition of binding was calculated using the formula${\% \mspace{14mu} {inhibition}}\; = {100 - \left( {\frac{A}{B} \times 100} \right)}$where A = binding in the presence of test serum; B = mean binding in thepresence of a pool of healthy blood donor sera

TABLE 8 Effect of patient sera on binding of the TSHR to 16E5 F(ab)₂coated tubes ¹²⁵I-43E1 labelled TSHR Inhibition Test bound to 16E5F(ab)₂ coated of Inhibition of TSH material¹ tubes (% total countsadded) binding² binding (%)³ G43 1.8 91.4 72.3 G44 4.8 77.2 45.1 G45 3.085.6 71.8 G46 2.0 90.2 83.8 G47 1.8 91.4 75.3 NSF 10 17.8 14.4 <14 NSF17 14.8 29.1 <14 NSF 21 13.5 34.9 <14 Table 8 footnotes: ¹Sera G43-47are from patients wwith Graves' disease; Sera NSF are from healthy blooddonors ²Inhibition of binding was calculated using the formula:${\% \mspace{14mu} {inhibition}}\; = {100 - \left( {\frac{A}{B} \times 100} \right)}$where A = binding 4E31 binding in the presence of test serum; B = meanlabelled 4E31 binding for healthy blood donor sera (15.4%) ³Inhibitionof TSH binding >14% is positive; PEG method used

TABLE 9 Effect of patient sera on binding of the TSHR to 14D3 F(ab)₂coated tubes ¹²⁵I-4E31 labelled TSHR Inhibition Test bound to 14D3F(ab)₂ coated of Inhibition of TSH material¹ tubes (% total countsadded) binding² binding (%)³ Serum A 4.0 70 72 Serum B 6.9 49 40 Serum C3.0 78 85 Serum D 2.6 81 80 NSF 5 15.1 −12 <14 NSF 17 14.6 −9 <14 NSF 2112.0 10 <14 NSF 23 11.8 12 <14 Table 9 footnotes: ¹Sera A-D are frompatients with Graves' disease; Sera NSF are from healthy blood donors²Inhibition of binding was calculated using the formula:${\% \mspace{14mu} {inhibition}}\; = {100 - \left( {\frac{A}{B} \times 100} \right)}$where A = labelled 4E31 binding in the presence of test serum; B = meanlabelled 4E31 binding for healthy blood donor sera (15.4%) ³Inhibitionof TSH binding >14% is positive; PEG method used

TABLE 10 Inhibition of ¹²⁵I-16E5 Fab binding to TSHR by TSHR MAbs IgG(100 μg/ml) % inhibition Epitope region (aa) 16E5 70.4 — 17D3 68.8 —14D3 67.6 — 17D2 69.2 — 2G2 −ve Thyroglobulin specific 5D6 −ve 22-41 8E2−ve 22-41 4B5 7.1 22-41 10C4 −ve 37-56 10D5 −ve 37-71 4D2 −ve 37-71 2E2−ve 52-71 1D6 −ve 202-221 7B5 −ve 202-221 16B6 −ve 202-221 3C3 11.2202-236 4B4 −ve 217-236 4E4 −ve 217-236 8D3 −ve 217-236 6D7 −ve 217-23618C5 15.7 246-260 3C7 17 246-260 4D7 24.2 246-260 3B3 8.8 277-296 5B5−ve 307-326 4E6 −ve 307-326 6E2 −ve 322-341 9C2 −ve 322-341 6B4 −ve337-356 3E4 −ve 337-371 3F3 −ve 352-371 3B2 −ve 352-371 7C2 −ve 367-3862B4 −ve 381-385 3E6 5.4 381-385 8E3 4.8 381-385 7C4 −ve 381-385 1D5 4.2381-385 4E2 −ve 381-385 3D3 −ve 382-401 2C4 −ve 382-401 10C2 −ve 382-4017E5 −ve 382-401

1-37. (canceled)
 38. A method of screening a sample of body fluid forTSH receptor autoantibodies wherein said sample is from a subjectsuspected of suffering from, susceptible to, having, or recovering fromautoimmune disease associated with an immune reaction to a TSH receptor,said method comprising: (a) contacting said sample with a polypeptidecomprising amino acid numbers 246-260 of a TSH receptor wherein thepolypeptide is not full length TSH receptor, so as to permit saidpolypeptide to bind with TSH receptor autoantibodies present in, orisolated from, said sample; and (b) detecting binding of saidpolypeptide with said autoantibodies thereby providing an indication ofthe presence of said autoantibodies in said sample, or isolated fromsaid sample.
 39. A method according to claim 38, of screening forautoantibodies produced in response to a TSH receptor in a sample ofbody fluid obtained from said subject.
 40. A method according to claim38, of screening for lymphocytes produced in response to a TSH receptorin a sample of body fluid obtained from said subject.
 41. A method ofscreening a sample of body fluid for TSH receptor autoantibodies whereinsaid sample is from a subject suspected of suffering from, susceptibleto, having, or recovering from autoimmune disease associated with animmune reaction to a TSH receptor, said method comprising: (a)contacting said sample with a polypeptide comprising amino acid numbers246-260 of a TSH receptor wherein the polypeptide is not full length TSHreceptor, so as to permit said polypeptide to bind with TSH receptorautoantibodies present in, or isolated from, said sample; and (b)detecting binding of said polypeptide with said autoantibodies therebyproviding an indication of the presence of said autoantibodies in saidsample, or isolated from said sample; which comprises directlymonitoring binding of autoantibodies to a TSH receptor present in thesample of body fluid from the subject and a polypeptide comprising aminoacid numbers 246 to 260 of a TSH receptor employing non-competitivesandwich type assay techniques.
 42. A method of screening a sample ofbody fluid for TSH receptor autoantibodies wherein said sample is from asubject suspected of suffering from, susceptible to, having orrecovering from autoimmune disease associated with an immune reaction toa TSH receptor, said method comprising: (a) contacting said sample witha polypeptide comprising amino acid numbers 246-260 of a TSH receptorwherein the polypeptide is not full length TSH receptor, so as to permitsaid polypeptide to bind with TSH receptor autoantibodies present in, orisolated from, said sample; and (b) detecting binding of saidpolypeptide with said autoantibodies thereby providing an indication ofthe presence of said autoantibodies in said sample, or isolated fromsaid sample; which employs at least one competitor capable of competingwith autoantibodies to a TSH receptor in the binding thereof with apolypeptide comprising amino acid numbers 246-260 of a TSH receptor. 43.A method according to claim 42, wherein said competitor comprises one ormore antibodies.
 44. A method of screening a sample of body fluid forTSH receptor autoantibodies wherein said sample is from a subjectsuspected of suffering from, susceptible to, having or recovering fromautoimmune disease associated with an immune reaction to a TSH receptor,said method comprising: (a) providing said sample of body fluid fromsaid subject; (b) contacting said sample with (i) a TSH receptorcomprising amino acid numbers 246-260, and (ii) at least one competitorcapable of competing with autoantibodies to a TSH receptor in thebinding thereof with a polypeptide comprising amino acid numbers 246-260of a TSH receptor, so as to permit said TSH receptor comprising aminoacid numbers 246-260 to bind with either autoantibodies to a TSHreceptor present in said sample, or said competitor; and (c) detectingthe binding of said TSH receptor comprising amino acid numbers 246 to260 with said autoantibodies present in said sample, thereby providingan indication of the presence of said autoantibodies to a TSH receptorin said sample. 45-51. (canceled)
 52. A method of diagnosing the likelyonset or presence of autoimmune disease associated with an immunereaction to a TSH receptor in a subject suspected of suffering from,susceptible to, having or recovering from, autoimmune disease associatedwith an immune reaction to a TSH receptor, the method comprisingdetecting autoantibodies produced in response to a TSH receptor in asample of body fluid from the subject according to claim 38, and wherebythe detected autoantibodies can provide a diagnosis of the likely onsetor presence of autoimmune disease associated with an immune reaction toa TSH receptor in the subject. 53-72. (canceled)
 73. A method ofscreening a sample of body fluid for autoantibodies to a TSH receptorwherein said sample is from a subject suspected of suffering from,susceptible to, having or recovering from autoimmune disease associatedwith an immune reaction to a TSH receptor, said method comprising: (a)contacting said sample with (i) a full length TSH receptor, one or moreepitopes thereof or a polypeptide comprising one or more epitopes of aTSH receptor, and (ii) one or more antibodies for a TSH receptor, whichantibody is capable of binding to a TSH receptor so as to stimulate theTSH receptor, which antibody does not comprise TSH or naturally producedautoantibodies to the TSH receptor; so as to permit said TSH receptor,said one or more epitopes thereof or said polypeptide, to bind witheither autoantibodies to a TSH receptor present in said sample, or saidone or more antibodies for the TSH receptor; and (b) detecting bindingof said TSH receptor, said one or more epitopes thereof or saidpolypeptide, with said autoantibodies present in said sample, therebyproviding an indication of the presence of said autoantibodies to a TSHreceptor in said sample.
 74. A method according to claim 73, whereinsaid polypeptide is a with which autoantibodies produced in response toa TSH receptor can bind and which comprises, consists of or consistsessentially of part or all of the primary structural conformation of oneor more epitopes of a TSH receptor with which autoantibodies produced inresponse to a TSH receptor can bind, which polypeptide comprises,consists of or consists essentially of the primary structuralconformation of one or more of the following: amino acid numbers 22 to91 of a TSH receptor; amino acid numbers 246 to 260 of a TSH receptor;amino acid numbers 260 to 363 of a TSH receptor; and amino acid numbers380 to 418 of a TSH receptor; with which autoantibodies produced inresponse to a TSH receptor can bind, with the exception of a full lengthTSH receptor.
 75. A method according to claim 73, wherein said one ormore epitopes of a TSH receptor with which autoantibodies produced inresponse to a TSH receptor interact, said one or more TSH receptorepitopes comprising, consisting of or consisting essentially of one ormore of the following: amino acid numbers 22 to 91 of a TSH receptor;amino acid numbers 246 to 260 of a TSH receptor; amino acid numbers 260to 363 of a TSH receptor; and amino acid numbers 380 to 418 of a TSHreceptor.
 76. A method according to claim 73, which comprises providinglabeling means for said one or more antibodies for a TSH receptor, whichantibody is capable of binding to a TSH receptor so as to stimulate theTSH receptor, which antibody does not comprise TSH or naturally producedautoantibodies to the TSH receptor.
 77. A method of screening a sampleof body fluid for TSH receptor autoantibodies wherein said sample isfrom a subject suspected of suffering from, susceptible to, having orrecovering from autoimmune disease associated with an immune reaction toa TSH receptor, said method comprising: (a) contacting said sample with(i) a full length TSH receptor, one or more epitopes thereof or apolypeptide comprising one or more epitopes of a TSH receptor, and (ii)one or more binding members for a TSH receptor; so as to permit said TSHreceptor, said one or more epitopes thereof or said polypeptide, to bindwith either autoantibodies to a TSH receptor present in said sample, orsaid one or more binding members; and (b) detecting binding of said TSHreceptor, said one or more epitopes thereof or said polypeptide, withsaid autoantibodies present in said sample, thereby providing anindication of the presence of said autoantibodies to a TSH receptor insaid sample; wherein said one or more binding members are directly orindirectly immobilised to a surface either prior to, or after step (a).78. A method according to claim 77, wherein said binding membercomprises a antibody for a TSH receptor, which antibody is capable ofbinding to a TSH receptor so as to stimulate the TSH receptor, whichantibody does not comprise TSH or naturally produced autoantibodies tothe TSH receptor.
 79. A method according to claim 77, wherein saidpolypeptide is a polypeptide with which autoantibodies produced inresponse to a TSH receptor can bind and which comprises, consists of orconsists essentially of part or all of the primary structuralconformation of one or more epitopes of a TSH receptor with whichautoantibodies produced in response to a TSH receptor can bind, whichpolypeptide comprises, consists of or consists essentially of theprimary structural conformation of one or more of the following: aminoacid numbers 22 to 91 of a TSH receptor; amino acid numbers 246 to 260of a TSH receptor; amino acid numbers 260 to 363 of a TSH receptor; andamino acid numbers 380 to 418 of a TSH receptor; with whichautoantibodies produced in response to a TSH receptor can bind with theexception of a full length TSH receptor.
 80. A method according to claim77, wherein said one or more epitopes of a TSH receptor are epitopeswith which autoantibodies produced in response to a TSH receptor bind,said one or more TSH receptor epitopes comprising, consisting of orconsisting essentially of one or more of the following: amino acidnumbers 22 to 91 of a TSH receptor; amino acid numbers 246 to 260 of aTSH receptor; amino acid numbers 260 to 363 of a TSH receptor; and aminoacid numbers 380 to 418 of a TSH receptor.
 81. A method according toclaim 77, which comprises providing labeling means for said TSHreceptor, said one or more epitopes thereof or said polypeptide. 82-100.(canceled)